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Characterization of the physiological role of the Microsporum gypseum alkaline protease during macroconidium… Page, William James 1973

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CHARACTERIZATION OF THE PHYSIOLOGICAL ROLE OF THE MICROSPORIA GYPSEUM ALKALINE PROTEASE DURING MACROCONIDIUM GERMINATION AND OUTGROWTH by WILLIAM JAMES PAGE B.S c , University of B r i t i s h Columbia, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced, degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Botany  The University of British Columbia Vancouver 8 , Canada Date ABSTRACT The results of this investigation suggested that Microsporum gypseum macroconidium germination was initia t e d by an alkaline protease and that this enzyme's activity, and hence the number of spores in i t i a t e d to germinate, was regulated by inorganic phosphate. Inorganic phosphate altered the germination protease pH optimum from pH 9 to pH 7 and de-creased i t s molecular weight from 33,000 to 16,000. Phosphate acted as a hyperbolic competitive inhibitor of germination protease activity, resulting in decreased activity towards ethyl-ester and spore coat substrates, and increased activity towards keratin substrates. The alkaline protease was inserted into the maturing spore coat as lysosomal vesicles, which upon spore rehydration, released their l y t i c contents into the cell wall. Owing to the complex construction of the fungal cell wall, the sequential action of other hydrolases was necessary to ensure sufficient spore coat lysis for germling outgrowth. Germination i n i t i a t i o n also involved the action of 81,3 glucanase, followed by chitinase, phosphodiesterase, and ethyl-esterase a c t i v i t i e s . The phosphodiesterase, which also was lysosomal, released spore coat phosphates for subsequent protease inactivation. Spore coat glycoproteins, extractable with ethylene diamine, were examined as possible germination protease substrates. A water-insoluble glycoprotein, which accounted for 10% of the spore coat dry weight and was modified in i t s total protein and amino acid content during sporulation, possibly served as the germination protease substrate i n vivo. The alkaline i i i protease also functioned i n the turnover of a water-soluble glycoprotein, which was not involved with sporulation, i n terminal stages of mycelial s t a r v a t i o n . Spore coat phosphates were determined to be located i n t e r n a l l y as phosphodiesters, and e x t e r n a l l y as exposed phosphate groups. These phosphate groups were deemed the possible s i t e s of phosphodiesterase action, as the phosphate content of both sources decreased during spore germination. The degree of a l k a l i n e protease i n a c t i v a t i o n by inorganic phosphate was found to be dependent on the r a t i o of phosphate to protease p r o t e i n . For example, i n a high density of spores the r a t i o of phosphate to protease p r o t e i n increased two-fold and the a c t i v i t y of the a l k a l i n e protease de-creased s t o i c h i o m e t r i c a l l y . By t h i s mechanism, a constant number of spores were germinated i n any given population. Calcium ions also were released p r i o r to germination i n i t i a t i o n . These ions possibly p r e c i p i t a t e d phosphates washed from the spores, thus preventing premature germination protease i n a c t i v a t i o n . Immediately a f t e r release from the fungal spore, the germination protease was a c t i v e against the outer k e r a t i n i z e d protein of h a i r ( 3 k e r a t i n ) . A f t e r phosphate-treatment, the protease also hydrolysed the i n t e r n a l f i b r i l l a r k e r a t i n of h a i r (a k e r a t i n ) . Germination protease a c t i v i t y towards k e r a t i n also was enhanced by d i s u l f i d e reducing agents and by k e r a t i n degradation products. The r e s u l t s suggested that the germination protease was con-verted i n t o a fu n c t i o n a l keratinase, immediately a f t e r germ tube emergence from the spore, thus ensuring the p a r a s i t i c s u r v i v a l of t h i s fungus on k e r a t i n i z e d t i s s u e s . M. gypseum sporulation was i n i t i a t e d by the hyphal t i p a f t e r i t s emergence from a submerged to a e r i a l environment. Reduction of a e r i a l environment humidity was implicated as the sporulation inducing f a c t o r . Forced dehydration by increased aeration caused a normally asporogenous pleomorphic s t r a i n of M. gypseum to sporulate. The induced wild-type s t r a i n c h a r a c t e r i s t i c s , however, were s t r i c t l y phenotypic and r e v e r s i b l e . Other pleomorphic s t r a i n s of d i f f e r e n t dermatophyte species s i m i l a r l y were induced to sporulate by increased aeration, suggesting that the pleomorphic variant r e s u l t e d from a common a l t e r a t i o n . TABLE OF CONTENTS Page General Introduction. 1 Text I. Regulation and s e l f - i n h i b i t i o n of Microsporum gypseum macroconidia germination. A. Introduction 3 B. Materials and Methods 4 C. Results 6 D. Discussion 20 I I . I s o l a t i o n and ch a r a c t e r i z a t i o n of M. gypseum lysosomes: r o l e of lysosomes i n macroconidia germination. A. Introduction 22 B. Materials and Methods 23 C. Results 27 D. Discussion 42 I I I . I n i t i a t i o n of dermatophyte pleomorphic s t r a i n sporulation by increased aeration. A. Introduction 46 B. Materials and Methods 47 C. Results 52 D. Discussion 64 IV. I s o l a t i o n and char a c t e r i z a t i o n of M. gypseum spore coat and mycelial wall glycoproteins: involvement i n spore germination. A. Introduction 68 B. Materials and Methods 70 C. Results 76 D. Discussion 99 v i TABLE OF CONTENTS (cont.) Page V. A l t e r a t i o n of the M. gypseum germination protease physical and p h y s i o l o g i c a l properties by inorganic phosphate: enhanced k e r a t i n o l y t i c a c t i v i t y . A. Introduction 107 B. Materials and Methods 109 C. Results 114 D. Discussion 150 General Discussion 160 Bibliography 166 LIST OF TABLES Table 1. Ratio of inorganic phosphate to protease protein i n a low spore-density germination system. Table 2. Stoichiometric i n h i b i t i o n of protease a c t i v i t y by inorganic phosphate. Table 3. Ratio of inorganic phosphate to protease protein i n low and high spore-density germination systems. Table 4. Comparison of co n t r o l and s t r a i n B17 inorganic phosphate to protease protein r a t i o s during low-density spore system germination. Table 5. E f f e c t of the presence of calcium ions on germination protease s p e c i f i c a c t i v i t y . Table 6. L o c a l i z a t i o n of the a l k a l i n e protease i n spore coat m a t e r i a l . Table 7. Binding of a l k a l i n e protease with spore coat material before and a f t e r spore germination. Table 8. S t a b i l i t y of the a l k a l i n e protease-binding with the spore coat. Table 9. Enzymes present i n i s o l a t e d v e s i c l e s . Table 10. Comparison of d i f f e r e n t pleomorphic species' response to aeration. Table 11. D i s t r i b u t i o n of protein i n i s o l a t e d mycelial walls and spore coats. Table 12. Percent of unfractionated c e l l wall dry weight that i s ethylene diamine extractable glycoprotein. Table 13. Comparison of amino acid and amino sugar compositions of glycoproteins from asporogenous hyphae and from macroconidia. Table 14. Comparison of amino acid and amino sugar composition of glycoprotein r e l a t i v e to l y s i n e content. Table 15. Comparison of Fraction A and B glycoproteins as a l k a l i n e protease substrates. Table 16. Hydrolase a c t i v i t i e s i n mycelial and germinating spore f r a c t i o n s . v i i Page 11 12 14 16 19 32 33 34 38 63 78 80 85 86 90 92 LIST OF TABLES (cont.) Table 17. C e l l w all and spore coat linkage content. Table 18. Keratin e x t r a c t i o n procedure. Table 19. Comparison of i n t r a c e l l u l a r and e x t r a c e l l u l a r a l k a l i n e protease s p e c i f i c i t y for substrate. Table 20. Relative a c t i v i t y of 10 hr germination protease on k e r a t i n f r a c t i o n s . Table 21. Molecular species of a l k a l i n e protease during sp o r u l a t i o n . Table 22. Percent u t i l i z a t i o n of k e r a t i n sources by s t r a i n s of M. gypseum. Table 23. E f f e c t of storage at -10 C on the molecular weight and enzyme a c t i v i t y of the large molecular weight keratinase. Table 24. E f f e c t of the method of s t e r i l i z a t i o n on the percent u t i l i z a t i o n of chicken feather k e r a t i n . Table 25. Comparison of the e f f e c t of cations on the a keratinase and a l k a l i n e protease a c t i v i t y of the 16,000 MW keratinase. Table 26. Evidence f o r isologous and heterologous as s o c i a t i o n of the keratinase subunits. v i i i Page 96 111 116 117 123 125 131 133 149 155 IX LIST OF FIGURES Page F i g . 1. M. gypseum germination protease molecular weight estimation. 7 F i g . 2. Release of protease protein and inorganic phosphate i n t o the germination f l u i d during low density macroconidium germination. 8 F i g . 3. Germination percentages of the low spore-density systems. 9 Fi g . 4. Release of protease protein and inorganic phosphate in t o the germination f l u i d i n low and high spore-density systems. 10 F i g . 5. I n a c t i v a t i o n of p u r i f i e d protease s p e c i f i c a c t i v i t y with inorganic phosphate. 15 F i g . 6. E f f e c t of calcium ions on protease a c t i v i t y and microscopic morphology of s t r a i n B17 germinating macroconidia. 17 F i g . 7. Comparison of s t r a i n R87 CFX protease and germination protease a c t i v i t y versus pH. 28 F i g . 8. Presence of acid and a l k a l i n e protease a c t i v i t y i n the CFX of s t r a i n R87. 29 F i g . 9. Changes i n protease enzyme a c t i v i t y during growth. 30 F i g . 10. Separation of acid and a l k a l i n e protease a c t i v i t i e s by f l o t a t i o n i n the presence of F i c o l l . 35 F i g . 11. Separation of acid and a l k a l i n e protease a c t i v i t i e s by sedimentation on a F i c o l l gradient. 37 F i g . 12. Cross-sectional view of a macroconidium a f t e r 2 hr germination. 39 F i g . 13. Longitudinal view of a macroconidium a f t e r 4 hr germination. 40 F i g . 14. Longitudinal view of a macroconidium a p i c a l t i p a f t e r 7 hr i n germination medium. 41 Fi g . 15. Proposed schematic port r a y a l of the o r i g i n s , d i s t r i b u t i o n and function of germination lysosomes i n M. gypseum. 45 F i g . 16. Apparatus used for the aeration of pleomorphic s t r a i n s . 49 LIST OF FIGURES (cont.) Page Fig. 17. Microscopic appearance and diagrammatic representation of developing strain R87P1 macroconidia. 51 Fig. 18. Comparison of strains R87 and R87P1 dry weight and percent intracellular water on cellophane-grown cultures. 53 Fig. 19. Early induction of strain R87P1 sporulation by aeration of pregrown colonies. 54 Fig. 20. Variable sporulation response of 4-day pregrown strain R87P1 to different air flow rates. 55 Fig. 21. Comparison of dry weight and appearance of macro-conidia in aerated and nonaerated strain R87P1 colonies. 57 Fig. 22. Optimum nutrient concentration for strain R87 and strain R87P1 sporulation. 58 Fig. 23. Formation of macroconidia after shift-down of strain R87 or strain R87P1 from complete medium to minimal agar medium. 60 Fig. 24. Comparison of c e l l walls and spore coats as alkaline protease substrates. 77 Fig. 25. Congruent electrophoresis of PAS and Coomassie blue staining bands from glycoprotein preparations. 79 Fig. 26. Changes in glycoprotein protein: hexose ratios during sporulation. 81 Fig. 27. Electrophoretic mobilities of glycoprotein Fractions A. 83 Fig. 28. Different electrophoretic mobilities of glyco-protein Fractions B. 84 Fig. 29. Effluent peaks from the short column for basic amino acids using strain R87P1 Fraction A glyco-protein hydrolysate. 87 Fig. 30. Changes in the glycoprotein protein: hexose ratio with starvation. 91 Fig. 31. Incorporation of acid-insoluble phosphate into spore coats during sporulation. 94 xi LIST OF FIGURES (cont.) Page Fig. 32. First differential plot of the results of titration of strain R87 macroconidia. 95 Fig. 33. Pigment release during strain R87 macroconidium germination. 98 Fig. 34. Alteration in the germination protease pH optimum on casein in the presence of inorganic phosphate. 115 Fig. 35. A. Inhibition of 3 hr germination protease alkaline protease activity and concomitant keratinase activation. 118 B. Inhibition of alkaline protease BTEE esterase activity with inorganic phosphate and PMSF. 118 Fig. 36. pH activity profiles of the germination protease and keratinase In Veronal and phosphate buffers on a keratin substrate. 119 Fig. 37. pH activity profiles of the germination protease and keratinase in Veronal and phosphate buffers on 3 keratin substrate. 120 Fig. 38. Elution of the 3 hr germination protease from a G100 column with (A) Veronal buffer and (B) phos-phate buffer. 122 Fig. 39. Elution of the extracellular keratinase from a G100 column with phosphate buffer after concentration by (A) flash evaporation and (B) PEG or F i c o l l dialysis. 126 Fig. 40. Comparison of the (A) intracellular alkaline protease activity and the (B) intracellular keratinase activity in strains R87 and R87P1. 127 Fig. 41. Aggregation of the alkaline protease and keratinase after rechromatography of isolated keratinase fractions of molecular weight (A) 65,000, (B) 33,000, and (C) 16,000. 129 Fig. 42. Increase in the caseinolytic activity of the large molecular weight keratinase after storage at -10 C and repeated freeze-thaw. 130 Fig. 43. Comparison of the effect of dilution on keratinase activity i n the (A) absence and (B) presence of autoclaved feather solubles. 134 LIST OF FIGURES (cont.) Fig. 44. Comparison of the effect of dilution of the keratinase in the presence of 5mM (A) s u l f i t e and b i s u l f i t e , (B) dithiothreitol, and (C) cysteine. Fig. 45. Elution profiles of the keratinase from a G100 column after dialysis concentration against (A) PEG, (B) PEG + AFS, (C) PEG + 5mM cysteine, and (D) PEG + 5mM DTT. Fig. 46. Comparison of the elution of the 16,000 MW phosphate-treated germination protease after (A) reduction with 5mM DTT, then dialysis concentration against PEG, and (B) reduction with 5mM DTT, but without dialysis concentration. Fig. 47. Sigmoid kinetics of the 3 hr germination protease on denatured albumin substrate. Fig. 48. A double reciprocal plot of the rate of product formation by the 3 hr germination protease in the absence and presence of phosphate with denatured albumin substrate. Fig. 49. Plot of 1/v against inhibitor concentration showing hyperbolic competitive inhibition of the 3 hr germination protease by inorganic phosphate. Fig. 50. Rate plot of the effect of inorganic phosphate on the 3 hr germination protease on lysozyme substrate. Fig. 51. Comparison of the rate of lysozyme substrate ut i l i z a t i o n by the keratinase and the 3 hr germ-ination protease + inorganic phosphate. Fig. 52. Comparison of the sensitivity of the keratinase and the germination protease to urea inactivation. Fig. 53. Comparison of the effect of metal ions on germ-ination protease activity. Fig. 54. Summary of the proposed M. gypseum protease-keratinase molecular interconversions. x i i Page 135 137 138 140 141 142 143 145 146 147 153 Acknowledgements I wish to sincerely thank Dr. John Stock, my research supervisor, for his guidance and encouragement during the course of my research and in the prepar-ation of this thesis. I am indebted also to Dr. Barbara D i l l , for her con-structive criticism and knowledgeable assistance. The good counsel of Dr. C. Person is also appreciated. I also would like to thank Louise Avent for excellent technical assistance, Dr. Ian Taylor for assistance with the amino acid analyses, and Teresa Walters for assistance with the electron microscope. I also wish to thank Dr. Ian Taylor, Dr. Delpha Syecklocha, and Dr. Jim Hudson for the use of their equip-ment and their interest in aspects of this project. Thanks also go to Pat Kong, for the rough typing of this manuscript. General Introduction Pathogenic fungi, like Microsporum gypseum produce asexual, multinucleate spores called macroconidia when grown on solid substrates. These macroconidia do not possess the heat resistance and survival characteristics of bacterial endospores, nor do they possess much increased thermal resistance over that of vegetative mycelia (Sussman & Halvorson, 1966). Macroconidia contain abundant endogenous reserves and are capable of rapid germination as soon as they are introduced into a suitable medium (Leighton, Stock & Kelln, 1970; D i l l , Leighton & Stock, 1972). It i s , therefore, most likely that Microsporum gypseum macro-conidia are primarily intended for the dispersal of this organism. M. gypseum is geophilic, being found primarily i n the soils of North America and Europe. In this environment, the mold's a b i l i t y to form airborne spores and u t i l i z e a wide variety of complex substrates (Ito & Fu j i , 1958) may help ensure survival. Another common habitat of M. gypseum is the body hair of animals like guinea pigs, cats, dogs, horses, and the hair of human heads, causing a parasitic ringworm infection. In order to ensure survival in this environment, the dermatophyte spore must be able to germinate, penetrate and u t i l i z e native keratin proteins as sole carbon and nitrogen sources (Kunert, 1972). It was previously reported by Leighton & Stock (1970a) that M. gypseum macroconidia germination was mediated by an alkaline protease, apparently present in the spore coats and released on germination. This enzyme was shown to be the only l y t i c enzyme released during germination and i t could be purified easily from the spore germination supernatant f l u i d . Several reviews which have sug-gested the complexity of the structure and composition of fungal c e l l walls (Bartnicki-Garcia, 1968; Hunsley & Burnett, 1970; Necas, 1971). It seemed unlikely, therefore, that M. gypseum macroconidia germination was caused solely by simple proteolysis of the spore coat. 2 To understand the events regulating M. gypseum spore germination, some knowledge of the macroconidium structure i s necessary. Information was avail-able on Microsporum sp. c e l l wall composition from digest studies (Tomomatsu, 1961; Grappel et a l , 1969) and total composition data was also available (Leighton & Stock, 1970a). Electron micrographs showing the appearance of c e l l wall layers also have been published (Werner et a l , 1966; 1968). Under-standing apical growth (Bartnicki-Garcia & Lippman, 1969) and a knowledge of the order of subapical polymerization of fungal wall constituents (Ainsworth & Sussman, 1966), may prove to be valuable in constructing models for spore coat synthesis. In this thesis, the physiological role of the alkaline germination protease was investigated further. It was deemed of particular interest to elucidate the role of this protease in the in i t i a t i o n and possible regulation of macro-conidial germination, the nature of the germination protease substrate within the spore coat, and the mechanism of germination protease release from the germinating spores. In an attempt to assess some of the differences between the "dermatophyte" spore coat and the mycelial wall, the nature of sporulation i n i t i a t i o n f i r s t was investigated, and a possible model for this differentiation process was proposed. A comparison of the composition and construction of the spore coat and the mycelial wall then was conducted to estimate some of the differences between these structures and possibly to identify a site of germin-ation protease specific activity. The additional role of the germination pro-tease, after germ tube emergence, as an extracellular keratinase also was investigated. 3 Section I Regulation and Self-Inhibition of Microsporum gypseum Macroconidia Germination Introduction: Regulation of mycelial density on complex and defined culture media is one means of conserving nutrients for better survival of the fungal population (Trinci, 1969). Survival would be ensured further by controlling spore germ-ination, thus restricting the number of proliferating areas. Self-inhibition of pycnidiospore germination at unfavorable temperatures was examined by Blakeman (1969), and a similar control has been shown to exist in Microsporum  gypseum macroconidial germination (Leighton & Stock, 1969). Regulation of germ-tube outgrowth under favorable germination conditions has not been described. Preliminary work by Leighton and Stock (1970a) showed that M. gypseum germ-tube outgrowth from macroconidia was initiated by the action of an alkaline protease. The results of this investigation show that the percentage of germination in a population of M. gypseum macroconidia is regulated by release of a specific inhibitor of alkaline protease. 4 Section I Materials and Methods; Organisms. A strain of M. gypseum (Bodin) Guiart and Grigorakis, 1928, originally obtained from F. Blank, Temple University, Philadelphia, Pa., was used in a l l studies of spore germination and inorganic phosphate inhibition. Studies of the effect of calcium on protease activity were carried out by using mutant strain B17 of the organism, previously described S P 3 - P i g + (Leighton & Stock, 1970b). Sporulation medium, macroconidial preparation, enzyme collection and  purification. Spore production and isolation were carried out as described previously (Leighton & Stock, 1969). Macroconidial germination was estimated microscopically (Leighton & Stock, 1969). Collection and purification of alkaline protease released on germination was carried out as reported previously (Leighton & Stock, 1970a). Germination medium and conditions. Macroconidia were germinated in physio-logical saline at pH 6.5 and 37 C in 125 ml Erlenmeyer flasks containing 10 ml of medium. The flasks were shaken at 125 rev/min in a R77 Metabolyte shaker water bath (New Brunswick Scientific Co., Inc., New Brunswick, N.J.). Analytical determinations. Protein estimations were made by the method of Lowry et a l (1951). Inorganic phosphate was determined by the method of Chen et a l (1956). Protease activity was measured by the method of McDonald and Chen (1965), with 2% casein in 0.05 M Veronal buffer (pH 8.5). Protease specific activity was measured as caseinolytic units per milligram of enzyme protein (1 unit equals the amount of enzyme which w i l l solubllize the equivalent of 1 ug of bovine serum albumin per min). Incubation time was 60 min at 37 C. Optical densities were measured on a Gilford model 2400 spectrophotemeter (Gilford Instrument Laboratories, Inc., Oberlin, 0.). 5 Disc gel electrophoresis and molecular weight estimation. Disc gel electrophoresis was at pH 9.1, under the procedure suggested for the Canalco (Rockville, Md.) model 6 system. Localization of the protease on the gel has been described (Leighton & Stock, 1970a). Molecular weight was estimated on sodium dodecyl sulfate (SDS)-acrylamide gels as recommended by Shapiro et a l , (1967). Gels were stained with 0.25% Coomassie blue, destained in 7% acetic acid, and scanned with a Gilford linear transport (model 2410) fit t e d to a model 2400 recording spectrophotometer. The 12.5 cm gels were scanned at 2 cm/min (560 nm). An aperture plate (0.1 x 2.36 cm) was employed to maintain band resolution. Chemicals. Crystalline bovine serum albumin, pepsin, and trypsin were obtained from Sigma Chemical Co., St. Louis, Mo. Lysozyme and l a c t i c dehydro-genase were obtained from Worthington Biochemical Corp., Freehold, N.J., and a-amylase was obtained from Sankyo Co. Ltd., Japan. A l l other chemicals were reagent grade and were purchased from Fisher Scientific Co., Vancouver, B.C., Canada. 6 Results: Germination system. To f a c i l i t a t e collection of higher yields of alkaline protease, large numbers of macroconidia were germinated in comparatively small volumes of germination medium. This approach resulted in less protease activity than would result from a lower spore density. At low spore density (3 * 10 5 macroconidia per ml of germination medium), the rate of release of protease into the supernatant f l u i d during germination was linear. Protease was the only protein released into the supernatant fl u i d during germination and migrated as a single band on disc gel electrophoresis. A molecular weight of approximately 30,000 was calculated from the mobility of the protease on SDS-acrylamide gels (Fig. 1). Protease specific activity did not increase with time, but the total activity was maximal at 2 hr , decreasing thereafter (Fig. 2). Approximately 50% of the spores germinated during the f i r s t 2 hr, but then the rate declined. Germination at high spore concentrations (1.5 * 10 6to 1.5 * 10 7 macroconidia per ml of germination medium) resulted in lower percentage of germination at 2 hr, although the total number of spores per ml germinated in both the high and low density systems was nearly constant (Fig. 3). The specific activity of the protease also decreased as the spore density increased from 3 * 10 5 to 1.5 x 10 7 macroconidia per ml of germination medium (Fig. 4). Inhibition of germination protease by inorganic phosphate. The question arose whether inorganic phosphate could be involved in inhibition of protease released on germination. Figure 2 shows that inorganic phosphate was released from the germinating macroconidia. The ratio of released inorganic phosphate to enzymic protein was minimal at 2 hr and doubled at 6 to 11 hr (Table 1). This ratio closely followed the pattern of protease specific activity: the two fold decrease in specific activity after 2 hr was accompanied by a twofold i n -crease in the inorganic phosphate to enzyme protein ratio (Table 2). 7 FIGURE 1 . Densitometrie t r a c i n g of Microsporum gypseum germination protease and molecular weight e s t i m a t i o n on sodium dodecyl s u l f a t e - a c r y l a m i d e g e l s . O r i g i n of the g e l (A) i s on the l e f t ; t r a c k i n g dye (TD) i s on the r i g h t . Molecular weight (B) was estimated by comparison of the r e l a t i v e m o b i l i t i e s of the protease p r o t e i n and standard p r o t e i n s , as d e t a i l e d by Shapiro et a l ( 1 9 6 7 ) . 8 FIGURE 2. Release of protease p r o t e i n (•) and inorganic phosphate ( O) i n t o germination supernatant f l u i d during low-density macro-c o n i d i a l germination (3 x 10 5 spores/ml). Protease s p e c i f i c a c t i v i t y ( © ) measured as c a s e i n o l y t i c units of enzyme protein/mg, where 1 unit equals the amount of enzyme which w i l l s o l u b i l i z e the equivalent of 1 ug of bovine serum albumin/min. Incubation time was 60 min at 37 C. 5 0 2 4 0 O f— < 2 2 3 0 or lxl 0 z 2 0 lxl U D C lxl °- 10 ! 1 5 1 "fe Q Ul 2 or lxl O tO Lxl 2.25 or O o_ 2.0 1.8 1.5 3X10 5 1.5x106 3x10 6 9X10 6 1.5X10 7 N U M B E R OF S P O R E S / M L FIGURE 3. Germination percentages of the low spore-density systems (3 * 10 s spores/ml) and of the high spore-density systems (1.5 x 10 to 1.5 * 10 7 spores/ml) at 2 hr, 37 C. 10 FIGURE 4. Release of protease protein cross-hatched (bar) and inorganic phosphate (dotted bar) into germination supernatant fl u i d from spores germinating in low (3 * 10 5 spores/ml) and high (1.5 * 105 to 1.5 x 10 7 spores/ml) spore-density systems at 2 hr, 37 C. Protease specific activity (solid bar) was measured as in Figure 2. 11 TABLE 1. Ratio of inorganic phosphate to protease protein in a low spore-density germination system Germina- Protease Inorganic tion protein* phosphate Inorganic phosphate/ time (umoles/ ^ mole/ml) protease protein (hr) ml) 0 0 0 0 2 1 0.0525 52.5 X 10" 3 4 1.33 0.117 87.9 X 10 - 3 6 1.67 0.203 121.5 X 10" 3 11 5.35 0.535 100 X 10" 3 * Calculated by using a molecular wight of 30,000 for the protease. TABLE 2. Stoichiometric i n h i b i t i o n of protease s p e c i f i c a c t i v i t y by inorganic phosphate* Spore ger-mination time (hr) Protease s p e c i f i c a c t i v i t y Inorganic phosphate/ protease protein Fold decrease i n protease s p e c i f i c a c t i v i t y Fold increase i n inorganic phosphate/ protease protein r a t i o 2 11.3 0.0525 0 0 6 and 11 5.3 0.1107 2.13 2.10 (avg) * Values derived from Table 1 and F i g . 2. 13 Figure 4 shows that, i n the high spore-density system, more inorganic phosphate than protease protein was released. This resulted in both a higher ratio of inorganic phosphate to enzyme protein at 2 hr in the high density spore systems than in the low density systems, and in early inactivation of protease activity (Table 3). From Fig. 5, i t was calculated that 3.4 * 10~6 M inorganic phosphate per microgram of enzyme protein was required to cause a 50% loss in protease activity in vitro. As shown in Fig. 2, a 2.34 x 10 M inorganic phosphate increase from 6 to 11 hr of spore germination resulted in a concomitant 31.7% decrease in pro-tease activity. From this observation, the calculated amount of inorganic phosphate required to cause 50% inactivation of protease enzyme (1 yg/ml) in vivo was also 3.4 * 10 - 6 M. Removal of inorganic phosphate by calcium ions. During spore germination, calcium ions were released into the supernatant fl u i d at 0 to 2 hr in micromolar concentrations (T.J. Leighton, unpublished data). To determine the effect of calcium ions on the germination protease and on the germinating spore, the germination-defective mutant strain B17 was used. This spore-forming mutant strain appears to have a normal protease (unpublished data), but releases excess inorganic phosphate during early periods of germination, resulting in early inactivation of protease activity (Table 4). Microscopic examination showed that, upon germination, spores of strain B17 did not form discrete germ tubes, but swelled by imbibition, formed surface blebs, and then lysed within 4 hr. If high concentrations of calcium ions were added to the system, no swelling occurred and germ tubes formed normally (Fig. 6). Conidial morphology was stabilized and swelling did not occur at 10"*1* and 10 - 5 M calcium ions; however, germ tubes were not formed. No lysis occurred at 6 hr in samples containing 10 - 1 to 10~5 M calcium ions. TABLE 3. Ratio of inorganic phosphate to protease protein i n low and high spore-density germination systems Protease _ . , . A Inorganic Inorganic phos-Spores/ml v. . . phosphate phate/enzyme , N (umole/ml) protein ml; 3 x 10s 1.5 * 106 3 x 106 1.5 x 107 1.33 1.66 3.0 6.3 0 0.055 0.105 0.222 0 33.1 x i o - 3 35.0 x i o - 3 35.0 x io" 3 * Calculated by using a molecular weight of 30,000 f o r the protease. 15 FIGURE 5. I n a c t i v a t i o n of p u r i f i e d protease s p e c i f i c a c t i v i t y (115 yg of protease protein/ml) by inorganic phosphate. Protease s p e c i f i c a c t i v i t y was measured as i n Figure 2. TABLE 4. Comparison of inorganic phosphate to protease protein r a t i o s during low-density spore system germination of s t r a i n B17 and control spores _ . Protease Inorganic Inorganic Germination ^ . . , , ^ , , ^ / Protease . p r o t e i n * phosphate phosphate/ . f, time / i i s p e c i x i c \ (umoles/ (umole/ protease . .^ (hr) I N i\ * i a c t i v i t y ml) ml) p r o t e i n J Control 0 0.166 0 0 22.5 2 4.0 0.00111 27 x l O " 3 47.5 4 5.83 0.0028 70 x 1 0 - 3 14.2 6 8.83 0.00555 68 x 10" 3 16.2 St r a i n B17 0 1.16 0 0 9.5 2 2.5 0.00111 44 x 10- 3 8.5 4 3.0 0.00389 129 x IO" 3 7.5 6 8.0 0.0139 174 x 10" 3 3.7 * Calculated by using a molecular weight of 30,000 f o r the protease. 37 S P O R E M O R P H O L O G Y > t - 4 X U < U LL) OL uO S2x < LD QC O 1x j — STABILIZATION of SHAPE - j GERM |-LYSIS-J TUBES~j I E 1 i 11 • • • I 10 10 10 1CT I ' CONTROL 1Q 1 -,o3 C A L C I U M ION CONCENTRAT ION ( M O L A R ) FIGURE 6. Effect of calcium ions on protease activity and microscopic morphology after germination of mutant strain B17 macroconidia in a low-density spore system (3 * 10 5 spores/ml) at 6 hr, 37 C. 18 It was found that addition of calcium ions in concentrations from 10-^ to IO - 8 M, to control enzymatic activity during germination, did not cause an i n -crease in enzyme activity. Preincubation of alkaline protease with IO - 2 M calcium ions and then with IO - 3 M inorganic phosphate caused a reduction in the inhibitory effect of inorganic phosphate (without calcium ions, 41.4% reduction in enzymatic activity; with calcium ions, 29.4% reduction i n enzymatic activity). When inorganic phosphate and calcium ions were added simultaneously, the i n -hibitory effect of inorganic phosphate was reduced, but not as effectively as in preincubation with calcium ions (32.8% reduction in enzymatic activity; see Table 5). TABLE 5. Effect of the presence of calcium ions on germination protease specific activity Protease Reduction Assay conditions specific in enzyme activity activity (%) Control 50.0 0 Preincubation, 10" •3 M phosphate 29.3 41.4 No preincubation, 10"3 M phosphate 30.0 40.0 Preincubation, 10" ' 2 M calcium ions* 35.3 29.4 Preincubation, 10" '** M calcium ions 33.3 33.4 No preincubation, IO - 2 M calcium ionst 33.6 32.8 * Molar concentrations of calcium ions were prepared from the chloride salt. The reaction mixture of enzyme and calcium ions was preincu-bated for 30 min at 37 C, and 10 - 3 M phosphate and 2% casein substrate (pH 8.5) were added. The reaction was continued for 60 min at 37 C. t Both iO""2 M calcium ions and 10 - 3 M phosphate were added to the react-ion mixture, and the reaction was continued for 60 min at 37 C. 20 Discussion: The concept of protease-mediated germination in M. gypseum was reported recently (Leighton & Stock, 1970a). Proteolysis has been related to germination of bacterial spores (Sierra, 1967; Strange and Dark, 1957) and fungal spores (Skucas, 1966; Hawker et a l , 1970). With fungal spores, the action of the protease may be on a protein "plug" in the spore coat (Skucas, 1966), or i t may be an overall hydrolysis and weakening of spore coat layers (Hawker et a l , 1970). M. gypseum macroconidia appear to be of the latter type, with the germ tube emerging from the most weakened area of the coat under the greatest hydrostatic pressure. Because the action of the protease appeared to produce a general, un-checked hydrolysis of spore coat proteins, this conceivably could lead to sphero-plast formation. However, as this was not observed, inactivation of the germination protease was believed to occur. The protease was inactivated after i n i t i a l germination (2 hr). Inorganic phosphate was released after this period, and protease activity decreased twofold. The relationship of phosphate to protease enzyme appeared to be stoichiometric; i.e., a twofold increase in the inorganic phosphate to protease protein ratio caused a twofold decrease in specific activity. This decreased activity re-sulted in a slower rate of macroconidial germination. In the high spore-density system, inorganic phosphate was released in excess of protease protein at early periods resulting in a lower percentage of germination. The total number of spores germinated, however, was the same, regardless of the spore density of the system. In this way, the number of germinating areas was restricted, allowing optimal growth of the fungal population. Phosphate is present in spore coats as phospholipids, hexose phosphates, and mannose 1,6 phosphodiester (Mill, 1966; Somers & Fisher, 1967; Fisher & Richmond, 1969). The mannose phosphate diesters are believed to be structural components of both yeast c e l l walls and fungal spore coats (Lampen, 1968). Thus, the action of a protease coupled with that of a 21 phosphodiesterase would weaken the spore coat for germ-tube outgrowth, causing the i n a c t i v a t i o n of the protease to occur through released inorganic phosphate. Although the protease can be completely i n h i b i t e d by addition of high con-centrations of inorganic phosphate to the p u r i f i e d enzyme, i t was evident that the protease was i n h i b i t e d by only 50% i n the low-density germination system. When germination percentage reached 99% at 11 hr, a c t i v e germination protease s t i l l was present i n the supernatant f l u i d . The e a r l y release of calcium ions from the spores may act as a protective mechanism, preventing premature i n a c t i v a t i o n of protease by early release of inorganic phosphate or by presence of phosphates i n the environment. Addition of calcium ions i n 10-fold excess of phosphate ions without preincubation did not cause s i g n i f i c a n t enzymatic protection, nor did preincubation of the enzyme with the calcium ions at a concentration lower than that of added phosphate ions. Enzymatic protection was obtained only when calcium ions were i n excess of phos-phate ions and were preincubated with the enzyme. This indicated that calcium ion release was a very early function i n germination and that phosphate had a greater a f f i n i t y for combination with the enzyme than with calcium ions. After 2 hr, the released phosphate was i n excess of the released calcium ions, and p a r t i a l protease i n a c t i v a t i o n ensued. Calcium ions appeared to possess some morphology-stabilizing properties, evidenced by t h e i r e f f e c t on s t r a i n B17. A s i m i l a r s t a b i l i z a t i o n of L a c t o b a c i l l u s morphology by calcium ions has been reported by Kojima et a l (1970). M. gypseum macroconidia germination can be described as a s e r i e s of events occurring during a short time i n t e r v a l i n the following sequence: ( i ) early release of calcium ions and protease protein; ( i i ) release of inorganic phos-phate, which i s then p r e c i p i t a t e d by calcium ions; ( i i i ) release of inorganic phosphate i n excess of released calcium ions ( a f t e r 2 h r ) ; and (iv) i n a c t i v a t i o n of protease by 50% (at 6 to 11 h r ) . 22 Section II I s o l a t i o n and Characterization of Microsporum gypseum Lysosomes: Role of Lysosomes i n Macroconidia Germination Introduction: The excretion of e x t r a c e l l u l a r enzymes (Matile et a l , 1965; 0'Sullivan & Mathison, 1971), synthesis of c e l l wall material (McClure et al., 1968; Grove & Bracker, 1970; Heath & Greenwood, 1970), a u t o l y s i s of Coprinus lagopus f r u i t bodies (Iten & Matile, 1970), and budding of yeast c e l l s (Moor, 1965) have been reported to be associated with v e s i c u l a r bodies of various types. The cyto-plasmic o r i g i n of these v e s i c l e s i s not e n t i r e l y c l e a r . Both the endoplasmic reticulum (Moor, 1967) and the Golgi apparatus (Kazama & A l d r i c h , 1972) have been implicated as possible s i t e s of o r i g i n . Lysosomes, a class of v e s i c l e s of wide d i s t r i b u t i o n characterized by t h e i r content of l y t i c enzymes (Matile & Wienken, 1967), have been reported to be very important i n fungal a u t o l y s i s and i n f e c t i o n processes (Wilson et a l , 1970; Wilson, 1973). In the dermatophyte, Microsporum gypseum, an a l k a l i n e protease has been shown to be necessary for macroconidia! germination (Leighton & Stock, 1970a; Section I ) . In ungerminated spores, t h i s protease i s associated with the spore coat (Leighton & Stock, 1970a) and, during spore germination, i t i s released as an e x t r a c e l l u l a r enzyme (Section I ) . In the studies presented here, the c e l l u l a r l o c a l i z a t i o n and v e s i c u l a r nature of the acid and a l k a l i n e p r o t e o l y t i c enzymes of M. gypseum were i n v e s t i g -ated. The possible cytoplasmic o r i g i n and probable function of these v e s i c l e s i n macroconidia! germination i s discussed. 23 Materials and Methods: A strain of M. gypseum (Bodin; Guiart and Grigorakis, 1928; designated strain R87) originally obtained from F. Blank, Temple University, Philadelphia, Pa., was used in these investigations. An asporogenous pleomorphic strain of R87, previously designated Sp2 Pig (Leighton & Stock, 1970b) also was used in these studies. Sporulation medium, macroconidia preparation, and germination. Spore production and isolation were carried out as previously described (Leighton & Stock, 1969). Liquid cultures were grown in a nutrient medium which consisted of 1% glucose (w/v), 1% neopeptone, (w/v; Difco), and d i s t i l l e d water (pH 6.4). Liquid cultures were aerated by shaking a 250 ml Erlenmeyer flask containing 100 ml of medium at 125 rev/min in a R77 Metabolyte shaker water bath (New Brunswick Scientific Co., New Brunswick, N.J.), at 25 C. Spore germination procedures have been described previously (Section I ) . Cell-free extract preparation and gradient centrifugation. Cell-free extracts (CFX) were prepared by the liquid nitrogen method of Bleyman and Woese (1969). Debris was removed from the liquid nitrogen homogenate by centrifug-ation at 10,000 x g for 10 min at 4 C. Extracts for enzyme determinations were buffered in 0.05 M tris(hydroxymethyl)-aminomethane (Tris)-hydrochloride (pH 7.4). Spore coat material was prepared for enzyme determinations by grinding isolated macroconidia in liquid nitrogen as described for the CFX preparations. Homogenization was carried to 95% breakage, as estimated microscopically. The 10,000 x g pellet was washed an additional eight times in physiological saline (pH 6.5) and resedimented between washes at 500 x g for 2 min at room temperature (25 C) in a c l i n i c a l centrifuge (Clay-Adams Co. Inc., New York). Extracts for ultracentrifugation also were broken in liquid nitrogen in the presence of a viscous grinding solution containing 0.5 M sucrose and 50% 24 glycerol (v/v) in 0.01 M Tris (pH 7.5). The homogenate was centrifuged at 290 x g for 10 min, and the resulting pellet was suspended in grinding solution and resedimented at 200 x g for 10 min. The supernatant fractions from both 200 x g centrifugations were pooled and centrifuged at 10,000 x g for 10 min in a Sorvall SS-1 centrifuge. The 10,000 x g supernatant fraction was centrifuged at 30,000 x g for 1 hr, and the resulting supernatant fraction was centrifuged at 40,000 x g for 2.5 hr in a Spinco model L ultracentrifuge (#30 rotor). A l l centrifugations were carried out at 4 C, and a l l pellets were suspended in 0.1 M Tris (pH 7.5) for enzyme determinations. Spore extracts for gradient ultracentrifugation were prepared i n liquid nitrogen in the presence of 0.05 M Tris (pH 7.5) containing 0.25 M sucrose. Debris was removed from the homogenate by centrifugation at 3,000 x g for 20 min at 4 C. Vesicles were isolated by the method of Matile and Wienken (1967), by flotation in the presence of 8% F i c o l l , (w/v); Pharmacia, Uppsala, Sweden). Vesicles also were isolated by a modification of the method of Brown (1968) by sedimentation of 5 ml of homogenate through a 20 ml linear 0 to 20% F i c o l l gradient containing 0.25 M sucrose and 0.05 M Tris (pH 7.5). The gradients were centrifuged at 10,000 rev/min for 15 min (flotation) or at 30,000 rev/min for 60 min (sedimentation) at 4 C in a Spinco model L ultracentrifuge (#30 rotor). Gradient fractions (1 ml) were collected with an Isco model A fraction collector (Instrument Specialties Co. Inc., Lincoln, Nebraska). Analytical determinations. Protein estimations in CFX and spore coat material were made by the method of Lowry et al (1951). Protein estimations in the presence of F i c o l l or sucrose were made after precipitation of the sample with 10% trichloroacetic acid and suspension of the pellet in 0.1 N sodium hydroxide. Relative protein concentrations in gradient fractions also were made by measuring absorbance at 280 nm. 25 Protease activity was measured by the method of McDonald and Chen (1965) with, 2% buffered casein. Acid protease determinations were made with 0.05 M citrate phosphate buffer (pH 5.0), and alkaline protease determinations were made with 0.05 M sodium barbital (Veronal) buffer (pH 9.0). Protease specific activity was measured as caseinolytic units per mg of enzyme or CFX protein, where one caseinolytic unit equals the amount of enzyme which w i l l cause an increased absorbance (280 nm) of 0.001 in acid-soluble material per min. Diaphorase and dihydronicotinamide adenine dinucleotide (NADH) reductase were measured by the method of Mahler by following the reduction of 2,6 dichloro-phenol-indophenol at 600 nm (Mahler, 1965). Phosphodiesterase was measured by the method of Neu and Heppel, employing bis-paranitrophenol phosphate as the substrate (Neu & Heppel, 1965), and acid phosphatase was determined by following the release of p-nitrophenol at 410 nm (Iten & Matile, 1970). A l l enzyme determinations were carried out at 37 C in a Gilford model 2400 recording spectrophotometer (Gilford Instrument Laboratories')'. Electron microscopy. Ungerminated and germinated spores were fixed in 1.5% (w/v) aqueous potassium permanganate, washed in 0.05 M sodium potassium phosphate buffer (pH 7.4) and then dehydrated in graded ethanol baths. Follow-ing infusion with propylene oxide, i n f i l t r a t i o n with Epon 812 was continued for 24 hr at 25 C. Samples were embedded in Epon 812 blocks containing 28.6 g of Epon 812, 21.0 g of dodecenyl succinic anhydride (DDSA), 10.53 g of nadic methyl anhydride (NMA), and 1% (w/v) DMP-30 catalyst. Polymerization was carried out at 37 C for 12 hr followed by 60 C for 30 hr. Blocks were sectioned with a LKB ultrotome (LKB-Produkter AB, Stockholm, Sweden). Sections were mounted on carbon-coated grids and post-stained with Reynold's lead citrate for 8 min and with 1% uranyl acetate for 30 min. Sections were scanned in a Phillips 300 electron microscope at 60 kv. 26 Chemicals. Substrates f or enzyme a n a l y s i s : paranitrophenol phosphate, bis-paranitrophenol phosphate, and 2,6 dichlorophenol indophenol were obtained from Calbiochem. NADH was obtained from Sigma Chemical Co. Reagents f o r electron microscopy: DDSA, NMA, and DMP-30 were obtained from Ernest F. Fullam, Inc. Epon 812, ethylenediaminetetraacetic a c i d , acid-washed casein, and a l l other reagent grade chemicals were obtained from Fisher S c i e n t i f i c Co., Ltd. (Vancouver, B.C.). 27 Results: Determination of the number of proteases present during sporulation. To determine the number of proteases active in sporulating M. gypseum, protease activity was determined over a wide range of pH values (Fig. 7). Comparison of the alkaline germination protease activity curve with the CFX protease activity curve showed that the CFX had more proteolytic activity at acidic pH levels than the germination enzyme, suggesting that there was an additional acid protease present in the CFX. Examination of the pleomorphic strain for the acid protease revealed a definite peak (Fig. 8), owing to the low alkaline protease levels in this mutant (Fig. 9). Treatment of sporulating strain R87 with phenyl methyl sulfonyl fluoride (a known inhibitor of the alkaline protease; Leighton & Stock, 1970a), allowed the detection of the acid protease at a level of activity comparable to the pleomorphic strain, suggesting that the acid protease was not involved directly with sporulation. Furthermore, acid protease activity i n -creased when the pleomorphic strain or strain R87 were grown in liquid culture on a complex nitrogen source (1% (w/v) casein), and acid protease activity de-creased when the strains were grown on a simple nitrogen source (1% (w/v) Casamino Acids). The alkaline protease activity level was unaffected by the complexity of the exogenous nitrogen source. These results suggested that the acid protease was assimilative and involved with mycelial growth. Occurrence of the alkaline protease during sporulation and germination. During sporulation of strain R87, alkaline protease levels increased, whereas in the asporogenous pleomorphic strain and the liquid-grown strain R87 (nonsporul-ating in liquid culture), the protease levels were much lower than those in the sporulating strain R87 (Fig. 9). The increase of 21 protease activity units from 4 to 5 days in sporulating strain R87 was entirely accounted for in the isolated spore CFX plus spore coat homogenate, and the apparent decrease of 17 28 T i i i i i r 4 5 6 7 8 9 10 11 p H FIGURE 7. Comparison of strain R87 CFX protease and germination protease activity versus pH. Specific activity = caseinolytic units per milligram of protein. (©) 5-day-old strain R87 sporulating culture CFX; ( O ) germination enzyme. 29 FIGURE 8. Presence of acid and alkaline protease in cell-free extract of strain R87. Specific activity = casein-olyt i c units per milligram of protein. (•) 5-day-old strain R87 sporulating culture CFX + IO - 3 M phenyl methyl sulfonyl fluoride (preincubated for 30 min at 37 C) ; (O) 5-day-old strain R87 sporulating culture CFX + 5 * 10 - 3 M phenyl methyl sulfonyl fluoride (pre-incubated for 30 min at 37 C); (•) 5-day-old pleo-morphic strain culture CFX. FIGURE 9. Changes i n protease enzyme a c t i v i t y during growth. S p e c i f i c a c t i v i t y = c a s e i n o l y t i c units per milligram of prot e i n . ( ® ) S t r a i n R87, sporul a t i n g culture ( s o l i d growth medium); (O) s t r a i n R87, nonsporulating culture ( l i q u i d growth medium); (•) pleomorphic s t r a i n ( s o l i d growth medium). 31 protease a c t i v i t y units from 5 to 7 days i n sporulating s t r a i n R87 was accounted f o r i n the spore coat material (Table 6). The protease a c t i v i t y associated with the spore coat material appeared as a decrease i n F i g . 9 since the spore coat material sedimented at 10,000 * g and was discarded ro u t i n e l y i n CFX preparation. S t a b i l i t y of the a l k a l i n e protease binding to the spore coat. Fractionation of ungerminated and germinated macroconidia by u l t r a c e n t r i f u g a t i o n (Table 7) con-firmed the previous observation of the association of the a l k a l i n e protease with spore coat material (Leighton & Stock, 1970a). In ungerminated spores, the protease i s t i g h t l y bound to the spore coat and l i t t l e a c t i v i t y was removed by washing with 0.05 M T r i s b u f f e r (pH 7.4; 200 x g wash supernatant f r a c t i o n ) . A f t e r germination and release of 56% of the protease into the germination medium, the remaining a c t i v i t y was le s s t i g h t l y bound to the spore coat and was removed i n the 200 * g wash supernatant f r a c t i o n (Table 7). Washing ungerminated spore coat material with various solvents indicated that the protease was not covalently bound to the spore coat since 97% of the a c t i v i t y was removed by washing with 8 M urea or with 10% sodium dodecyl s u l f a t e a f t e r 24 hr. The protease was u n l i k e l y to be associated with the spore coat as a free enzyme since i t was not t o t a l l y removed from the coats by any of the s o l -vents a f t e r 24 hr. The presence of 10 to 39% of the o r i g i n a l protease a c t i v i t y a f t e r 24 hr of washing with e i t h e r 0.05 M Veronal buffer or a s o l u t i o n with high s a l t concentration (6 M ammonium c h l o r i d e ) , r e s p e c t i v e l y , suggested that these conditions must s t a b i l i z e the binding of the protease to the spore coat (Table 8). Separation of acid and a l k a l i n e protease i n F i c o l l gradients. Acid protease-containing v e s i c l e s were demonstrable by the method of Matile and Wienken (1967), by f l o t a t i o n i n the presence of 8% F i c o l l ; however, by t h i s procedure, the a l k a l i n e protease a c t i v i t y sedimented with the spore coat material. As shown i n F i g . 10, the a l k a l i n e protease also was contained i n v e s i c l e s which could be TABLE 6. L o c a l i z a t i o n of the a l k a l i n e protease i n spore coat material CFX material Growth time (days) A l k a l i n e protease s p e c i f i c a c t i v i t y 3 Spores + mycelia^ 4-5 + 21 Spores + mycelia^ 5-7 - 17 Spore CFX + spore coats 7 22.5 Spore coats 7 15 Spore CFX 7 7.5 a In terms of c a s e i n o l y t i c units per milligram of protein at pH 9.0, 37 C; + indicates increased enzyme a c t i v i t y ; - i ndicates decreased enzyme a c t i v i t y . b Data derived from F i g . 9. TABLE 7. Binding of a l k a l i n e protease with spore coat material before and a f t e r spore germination Tot a l a l k a l i n e pro-tease u n i t s 3 (%) Fracti o n . . . _ Ungermi- Germi-nated nated spores spores 200 x g wash p e l l e t b 86.9 18.2 200 x g wash supernatant^ 3 f r a c t i o n 10.5 21.3 10,000 x g p e l l e t 0.4 2.6 30,000 x g p e l l e t 2.0 0.19 40,000 x g p e l l e t 0.0 7.7 40,000 x g supernatant f r a c t i o n 0.0 0.0 Germination medium 0.0 56.0 Calculated as = (units of protease a c t i v i t y per f r a c t i o n / u n i t s of protease a c t i v i t y per unfractionated homogenate) x 100%, where 1 unit of a c t i v i t y = (A OD 280 nm x 10 3) per min. P e l l e t (200 x g ) washed with 0.05 M T r i s b u f f e r (pH 7.4) and resedimented at 200 x g f o r 10 min. TABLE 8. S t a b i l i t y of the a l k a l i n e protease-binding with the spore coat Untreated spore coat a l k a l i n e protease S o l v e n t 3 s p e c i f i c a c t i v i t y b (%) A f t e r 24 hr A f t e r 48 hr of washing of washing 6 M NH^Cl 38.7 5.2 8 M Urea 2.4 0.0 10% Sodium dodecyl s u l f a t e 3.1 0.0 I O - 2 M Ethylenediamine-t e t r a a c e t i c a c i d 4.3 1.2 0.85% NaCI 5.3 0.0 0.85% NaCI (37 C) 4.4 0.0 0.05 M Veronal buffer (pH 9.0) 10.1 0.0 a A l l solvent washes c a r r i e d out at 25 C. k S p e c i f i c a c t i v i t y = units per milligram of spore coat material (dry weight). Unit = (A o p t i c a l density at 280 nm * 10 3) per minute. 35 FIGURE 10. Separation of acid and alkaline protease a c t i v i t i e s by flotation in the presence of F i c o l l . Specific activity = caseinolytic units per absorbance at 280 nm per fraction. (O) Acid protease activity; (©) alkaline protease activity. 36 separated from the acid protease-containing v e s i c l e s by f l o t a t i o n i n 0 to 20% F i c o l l gradients by the modified method of Brown (1968). Further c l a r i f i c a t i o n , of the protease a c t i v i t y bands was obtained by high-speed sedimentation (30,000 rev/min for 60 min at 4 C), through a 0 to 20% F i c o l l gradient. Under these conditions, the a c i d protease was present as one peak (A), and the a l k a l i n e protease was present i n two major peaks (B and C). The sedimentation of peak B suggested that these v e s i c l e s were very dense, whereas the peak C v e s i c l e s were more buoyant (Fig. 11). Enzymes associated with the acid and a l k a l i n e protease v e s i c l e s . Table 9 shows that maximal acid phosphatase a c t i v i t y was associated with the acid protease v e s i c l e s (peak A). The a l k a l i n e protease v e s i c l e s (peaks B and C) contained no acid protease a c t i v i t y and had less a c i d phosphatase a c t i v i t y than peak A. Phosphodiesterase a c t i v i t y was concentrated i n peak B with some a c t i v i t y present i n peak C. The f r a c t i o n s were free of mitochondrial contamination as judged by the absence of NADH reductase a c t i v i t y . Electron microscopic examination. The macroconidia of M. gypseum appeared multiseptate and elongate under examination with the l i g h t microscope. The germ tube arose terminally as noted previously (Section I ) . In the 2 hr germinating spore, t h i n cross sections revealed a thick spore coat of at l e a s t three lay e r s . Small v e s i c l e s were v i s i b l e i n s i d e the spore coat layer next to the plasmalemma (Fi g . 12). Spores which had been shaken for 4 hr i n germination medium had peri p h e r a l membrane-bound bodies which were e i t h e r large and electron-dense (DV) or small and electron-transparent (TV) v i s i b l e within the spore coat material. The inner l a y e r of the spore coat appeared less granular around the TV than around the DV (Fig. 13). Vesicles appearing i n spores germinated a f t e r 7 hr were predominantly of the large, electron-dense type and were seen within the areas of the inner spore coat which had become less granular (Fig. 13 and 14). PERCENT F ICOLL 20 15 10 5 0 i — i — i — i — i — i — i — i i — i i i i i i ' ' ' i i i i i i i 1 5 10 15 20 25 FRACT ION . N U M B E R FIGURE 11. Separation of acid and alkaline protease activities by sedimentation on a F i c o l l gradient. Specific activity = caseinolytic units per absorbance at 280 nm per fraction. (0;9) Duplicate experiments, protease ac t i v i t y , pH 9.0; (D;B) duplicate exper-ments, protease activity, pH 5.0. TABLE 9. Enzymes present i n i s o l a t e d v e s i c l e s Enzyme s p e c i f i c a c t i v i t y Determination Unfrac- „ . „ . „ , ^ , Peak Peak Peak tionated a a a CFX Al k a l i n e protease^ 76.28 9.25 34.41 154.78 Phosphodiesterase 0 64.39 0.0 114.28 37.01 Acid protease* 3 2.38 28.32 0.0 1.96 Acid phosphatase 0 29.77 177.61 70.93 57.40 Diaphorase^ 74.41 25.92 34.44 22.59 NADH reductase d 29.52 0.0 0.0 0.0 a Peaks A, B, and C of F i g . 11. k S p e c i f i c a c t i v i t y = c a s e i n o l y t i c units per milligram of pr o t e i n . c S p e c i f i c a c t i v i t y = [A o p t i c a l density (OD) at 410 nm * 10 3] per minute per milligram of prote i n . ^ S p e c i f i c a c t i v i t y = (A ODgoo x 102) per minute per milligram of protei n . FIGURE 12 . Cross-sectional view of a macroconidium a f t e r 2-hr germination. The spore coat i s composed of three l a y e r s : an inner granular layer (IW), an outer lamellar layer (OW), and an outer electron-dense layer (ODL). Numerous peripheral v e s i c l e s are located within the inner wall layer and are e i t h e r small electron-transparent v e s i c l e s (TV) or large - electron-dense v e s i c l e s (DV). The inner wall layer i s i n close contact (arrows) with the plasmalemma (P), where the v e s i c l e s are absent. 40 FIGURE 13. Longitudinal view of a macroconidium a f t e r 4 hr of germination. Both electron-transparent v e s i c l e s (TV), and electron-dense v e s i c l e s (DV) are seen within the spore coat inner w a l l (IW). The inner wall has reduced granularity (RG) around the electron transparent v e s i c l e s . 41 FIGURE 14. Longitudinal view of a germinating macroconidium apical tip after 7 hr in germination medium. The spore coat inner wall (IW) granularity i s reduced (RG) around the cytoplasmic material (CYT) and extends into the inner wall material. The apical vesicle (DV) is membrane-bound (M) and contains electron-dense material. 42 Discussion: The r e s u l t s of t h i s i n v e s t i g a t i o n have shown that M« gypseum mycelia con-tained two cytoplasmic proteases. During sporulation, an a l k a l i n e protease was incorporated i n t o the spore coat. The a l k a l i n e protease was not associated with the spore coat as a soluble or covalently bound enzyme but was l o c a l i z e d i n v e s i c l e s . These v e s i c l e s were seen i n electron micrographs of germinating M. gypseum macroconidia and were i s o l a t e d by F i c o l l gradient c e n t r i f u g a t i o n . Protease containing v e s i c l e s have been i s o l a t e d s i m i l a r l y from the dermatophyte M. canis by 0'Sullivan and Mathison (1971). Morphologically, the a l k a l i n e protease-containing v e s i c l e s resembled those i s o l a t e d from Neurospora crassa. where the protease-containing v e s i c l e s cross the plasmalemma as i n t a c t p a r t i c l e s by possible invagination of the plasmalemma (Matile et a l , 1965; Gibson & Peberdy, 1972). During M. gypseum macroconidial germination, the v e s i c l e s , which probably contained the a l k a l i n e protease, appeared electron transparent, and the spore coat around them became less gran-u l a r , probably due to p r o t e o l y s i s by released a l k a l i n e protease. The spore coat layer most affe c t e d by hydrolysis was that next to the plasmalemma. This layer has been reported to be a peptido-glucan i n Saccharomyces cerevisiae (Kidby & Davies, 1970) or a peptido-glucan-chitin complex i n Candida u t i l i s (Novaes-Ledieu & Garcia-Mendoza, 1970) and A s p e r g i l l u s nidulans (Gibson & Peberty, 1972). Furthermore, the observation that phosphatase and phosphodiesterase a c t i v i t y was associated with the a l k a l i n e protease-containing v e s i c l e s confirmed an e a r l i e r hypothesis that the source of phosphate which i n a c t i v a t e d the a l k a l i n e protease a f t e r germination was spore coat phosphates or phosphodiesters (Section I ) . The acid protease-containing v e s i c l e was shown to be an a s s i m i l a t i v e l y s o -some. Acid protease a c t i v i t y increased as the exogenous nitrogen source became more complex, i n d i c a t i n g the l y t i c - a s s i m i l a t i v e r o l e of these v e s i c l e s . Acid 43 phosphatase activity was associated with the acid protease, as is true for other assimilative lysosomes (Matile & Wienken, 1967; Brown, 1968). Also the be-haviour of the vesicles during isolation suggested that they were similar to assimilative lysosomes isolated from S. cerevisiae and rat livers (Matile & Wienken, 1967; Brown, 1968). The acid protease was not believed to be i n -volved in sporulation since i t was found at an equal level of activity in both the R87 strain during sporulation arid the asporogenous pleomorphic strain. The origin of the acid protease-containing vesicles from the endoplasmic reticulum (ER) was suggested by the presence of diaphorase activity (Marchant & Robards, 1968). The origin of the alkaline protease-containing vesicles was less obvious than that of the acid protease-containing vesicles. The presence of diaphorase activity suggested that the alkaline protease-containing vesicles also were de-rived from the ER. The distribution of alkaline protease and phosphodiesterase activity in peaks B and C (Fig. 11) suggested that the vesicles were of the same origin, possibly a multivesicular form of vesicle as suggested by Marchant and Robards (1968). These vesicles have appeared also i n Botrytis cinerea germ tubes (Gull & Tr i n c i , 1971) and in Crypto coccus neoformans bud areas (Takeo jet a l , 1973). It has been previously shown that during M. gypseum macroconidial germin-ation, alkaline protease was released from 0 to 2 hr with phosphate release and subsequent alkaline protease inactivation most pronounced from 5 to 8 hr (Section I). It has been shown here that the majority of the alkaline protease was associated with the spore coat prior to germination. These observations sug-gested that the buoyant vesicles (peak C), containing the most alkaline protease activity, were inserted into the spore coat prior to germination, and the denser vesicles (peak B), containing the greatest phosphodiesterase activity were 44 inserted into the spore coat after i n i t i a l germination and release of alkaline protease. This would permit spore coat hydrolysis to precede germination protease inactivation. Further evidence supporting this proposal was the observation of two types of vesicles within the inner spore coat layer. The electron transparent vesicles were the predominant type in spores germinated in 2 hr, with the DV appearing and becoming the predominant type in spores germinated 4 to 7 hr. The possible origins of the alkaline protease-containing vesicles are summarized in Fig. 15 where: (I) the alkaline protease-containing vesicles and, (II) the phosphodiesterase-containing vesicles were derived independently from the ER, or (III) the alkaline protease-containing vesicles and the phosphodiesterase-containing vesicles were derived from a common multi-vesicular body which had been formed directly from the ER. The latter inter-pretation was favoured since i t accounts for the presence of both phosphodiester-ase and alkaline protease activities i n both peaks B and C. The role of these lysosomes in macroconidial germination could, therefore, provide an ordered sequence of events: (1) insertion of alkaline protease-containing lysosomes into the spore coat prior to germination, (2) release of protease and proteolysis of the spore coat, (3) insertion of phosphodiesterase-containing vesicles into the spore coat, (4) release of phosphodiesterase, diester cleavage in the spore coat, and release of inorganic phosphate and, (5) phosphate inactivation of the alkaline protease in the germination medium. 45 P L A S M A L E M M A ! C O A T |~SPORE ' E N D O P L A S M I C R E T I C U L U M _ — f t A L K A L I N E P R O T E A S E ST\ mim _ I V E S I C L E S ^Jmm n ,o \MULT I V E S I C U L A R n\ B O D Y P H O S P H O D I E S T E R A S E . V E S I C L E S PRIOR TO G E R M I N A T I O N J A F T E R INITIAL G E R M I N A T I O N ( S U S P E C T E D ) FIGURE 15. Proposed schematic portrayal of the origins, distribution, and function of germination lysosomes of M. gypseum. (a) Inactivation of the germination protease by inorganic phosphate has been established previously (Section I ) . 46 Section I I I I n i t i a t i o n of Dermatophyte Pleomorphic S t r a i n Sporulation by Increased Aeration Introduction: Pleomorphism i n dermatophytes presents a problem i n both culture c o l l e c t i o n maintenance and the i d e n t i f i c a t i o n of these organisms. This spontaneous a l t e r -ation occurs at a high frequency i n many dermatophyte species when c l i n i c a l specimens are grown on a r t i f i c i a l c ulture media ( B i s t i s , 1960). The concomitant loss of wild-type pigmentation and spore-forming a b i l i t y makes generic and species i d e n t i f i c a t i o n impossible. Stimulation of sporulation of microconidiate pleomorphic s t r a i n s by use of increased carbon dioxide tension has been reported previously (Chin & Knight, 1957; Balabanoff, 1963). Induction of sporulation i n asporogenous pleomorphic s t r a i n s , however, has not been successful (Chin & Knight, 1957). Described here i s a new method f o r sporulation induction i n pleomorphic s t r a i n s , u t i l i z i n g c o n t r o l l e d aeration and dehydration. A possible explanation f o r the observed early induction of sporulation by aeration i s proposed and the re s u l t s of t h i s i n v e s t i g a t i o n suggest that further e l u c i d a t i o n of t h i s e f f e c t should prove to be important i n the i d e n t i f i c a t i o n of these organisms i n c l i n i c a l l a b o r a t o r i e s . Furthermore, the a b i l i t y to induce sporulation i n a normally asporogenous s t r a i n has proven very useful to accentuate sporulation s p e c i f i c processes and provides a pos s i b l e model for spore i n i t i a t i o n , development and maturation i n M. gypseum. 47 Materials and Methods; Organisms. A pleomorphic s t r a i n of Microsporum gypseum (R87P1), derived as a spontaneous a l t e r a t i o n of the wild-type s t r a i n (R87) previously described (Section I I ) , was used i n the majority of studies of growth and sporulation induction. Other pleomorphic s t r a i n s used i n t h i s study also were i s o l a t e d as spontaneously occurring pleomorphic patches within wild-type colonies which o r i g i n a l l y had been i s o l a t e d from c l i n i c a l specimens. A l l pleomorphic s t r a i n s were free of microconidia and macroconidia during t h e i r respective wild,-type s t r a i n sporulation cycles. Culture medium and aeration conditions. A l l c u l t u r e s , unless otherwise s p e c i f i e d , were c u l t i v a t e d on a medium containing: neopeptone (Dif c o ) , 1% (w/v); glucose, 1% (w/v); agar, 1.8% (w/v); and d i s t i l l e d water (pH 6.5). The medium was s t e r i l i z e d r o u t i n e l y by autoclaving at 121 C for 15 min except for the more concentrated types of media, which were s t e r i l i z e d by M i l l i p o r e f i l t r a t i o n . Culture f l u i d by-products were c o l l e c t e d from 7 day o l d liquid-grown cultures by f i l t r a t i o n , concentrated by f l a s h evaporation (27 C), and s t e r i l i z e d by M i l l i p o r e f i l t r a t i o n before a d d i t i o n to the culture medium as 0.5, 1.0, 5.0, and 7.5% supplements. The mycelial inoculum used was pregrown i n l i q u i d medium for 5 days at 27 C using a B u r r e l l wrist action shaker ( B u r r e l l Corp., Pittsburgh, Penn.). The mycelia then was c o l l e c t e d a s e p t i c a l l y by f i l t r a t i o n and washed with 250 ml of s t e r i l e p h y s i o l o g i c a l s a l i n e (pH 6.5). A mycelial inoculum which could be dispensed by pipette was prepared by shearing the washed hyphae i n a V i r T i s homogenizer (Model 23, V i r T i s Co. Inc., Gardiner, N.Y.) for 30 sec at approxi-mately 16,000 rev/min. A l l cultures were inoculated on agar media by using one drop of sheared mycelial suspension dispensed c e n t r a l l y onto the surface of the medium. 48 Cultures for n u t r i t i o n a l shift-down were grown i n l i q u i d medium described previously (Section I I ) . A f t e r growth for s p e c i f i c periods of time, the sub-merged mycelia were harvested a s e p t i c a l l y by f i l t r a t i o n and washed with 250 ml of s t e r i l e p h y s i o l o g i c a l s a l i n e , and the resultant mat was removed from the f i l t e r and placed i n a p e t r i p late containing 1.8% (w/v) agar (Difco) without a d d i t i o n a l n u t r i e n t s . Aerated cultures were grown i n 250 ml Erlenmeyer flasks containing 100 ml of agar medium. Glass tubing was arranged as shown i n F i g . 16A with the a i r i n l e t tube 1.5 cm above the growing colony. Cultures for dry weight and percentage water determinations were grown on the surface of 8.7 cm cellophane discs placed on deep p e t r i plates containing 70 ml of medium. Aeration of the p e t r i p late cultures was conducted i n the apparatus shown i n F i g . 1.6B, employing the glass tubing and stopper assembly depicted i n F i g . 16A, and enclosing the p e t r i p late culture i n a 16.51 by 20.96 cm p l a s t i c bag ("Baggie" brand, Colgate-Palmolive Ltd., Toronto, Canada). This method proved to be superior to the f l a s k method, both i n ease of sampling and for d i r e c t microscope observations. Aeration was supplied by a small aquarium pump (Tomofuji Co., Japan), and the a i r was s t e r i l i z e d by passage through a 17 by 2 cm tube containing s t e r i l e cotton wool. Dry or humid a i r was generated by d i r e c t i n g the airstream through a 17 by 2 cm tube containing s t e r i l e calcium c h l o r i d e , or a 250 ml Erlenmeyer f l a s k h a l f - f i l l e d with s t e r i l e d i s t i l l e d water. A i r flow was regul-ated by screw clamps and monitored with an RGI a i r - f l o w meter (Roger Gilmont Instruments, Inc., Great Neck, N.Y.). Incubation of fungal colonies under increased carbon dioxide tensions has been described previously (Chin & Knight, 1957). Growth measurement and c a l c u l a t i o n s . Growth was measured as dry weight, a f t e r desiccation i n vacuo over CaCl2 at 27 C, or as amount of hyphal extension 49 FIGURE 16. A and B, Apparatus used for aeration of pleomorphic strains. Details of construction and aeration methods used appear in Materials and Methods. 50 (radius [mm] per day). Dry weight of mycelia from solid culture media was estimated by using colonies grown on tared cellophane discs. The weight of intracellular water was estimated after the method of Ito and F u j i i (1958), as the difference in fungus weight after drying for 3 hr at 37 C and then after drying for 20 hr at 110 C. The percent intracellular water was calculated as weight of intracellular water divided by dry weight (37 C, 3 hr) times 100%. Estimation of spore induction and maturation. Spore formation studies followed the stages of development previously described for the wild-type strain by El-Ani (1963). Spore induction was designated as the time at which thin-walled, bulbous, aseptate macroconidia f i r s t appeared. Spore maturation was reported when the macroconidia became thick-walled, septate, and free from the vegetative hyphae (Fig. 17). Isolation of mature macroconidia and germination procedures have been described previously (Leighton & Stock, 1969). Photographs of lactophenol cotton blue-stained specimens were taken with a Zeiss microscope (green f i l t e r ) . •51 FIGURE 17. Microscopic appearance and diagrammatic representation of developing s t r a i n R87P1 macroconidia. Spore development proceeds clockwise from the uninduced pleomorphic hyphae. Spore i n i t i a t i o n f i r s t was marked by hyphal t i p swelling and formation of a basal septum. Further septation, d i -v i d i n g the swollen hyphal t i p into three compartments, and c e l l wall thickening marked the formation of immature spores. Mature spores were divided into at l e a s t four compartments and separated from the vegetative hyphae. 52 Results: Optimum ph y s i c a l conditions for pleomorphic s t r a i n sporulation. The pleo-morphic s t r a i n (R87P1) of M. gypseum was characterized by absence of reverse pigmentation, f l u f f y white mycelia, and reduced a b i l i t y to sporulate, with only very few macroconidia i n i t i a t e d a f t e r 12 days of growth. The wild-type s t r a i n (R87) was tan i n colour, granular, and produced abundant macroconidia a f t e r 4 days of growth (Fig. 18). Comparison of growth curves shows that s t r a i n R87 i n i t i a t e d sporulation p r i o r to rapid growth, whereas s t r a i n R87P1 i n i t i a t e d very sparse sporulation i n the stationary phase of growth. Despite the wide d i f f e r -ences i n sporulation times, there was an i d e n t i c a l percentage of i n t r a c e l l u l a r water loss at the time of mature spore appearance i n both s t r a i n s (Fig. 18). To determine the e f f e c t of aeration and drying on sporulation, the apparatus i n F i g . 16 was devised. Colonies of the pleomorphic s t r a i n were pregrown for 1 to 7 days ( i n 250 ml f l a s k s ) and aerated at 50 cm3/min to determine i f the colony could be stimulated to sporulate e a r l i e r than normal. As shown i n F i g . 19, s t r a i n R87P1 was induced to form spores by 8 days t o t a l growth, with optimal sporulation induction occurring when the colony was pregrown for 4 days. Aeration p r i o r to t h i s time or a f t e r i t increased the duration of the sporulation period. When colonies pregrown for 4 days were exposed to d i f f e r e n t a i r f l o w rates, optimum sporulation was obtained with a i r flow rates of 25 to 40 cm3/min (Fig. 20). A i r flow rates of 75 and 100 cm3/min gave slower spore induction but more rapid spore maturation. Aeration alone did not seem to be the cause of early sporulation induction, as humid aeration at 40 cm3/min did not stimulate sporulation as well as aeration at 40 cm3/min without a water trap. S i m i l a r l y , r apid dehydration (dry a i r at 40 cm3/min) did not stimulate induction of spores as well as 40 cm3/min of a i r flow rate under normal conditions, but i t did i n -crease rate of spore maturation. These r e s u l t s suggested that both c o n t r o l l e d 53 FIGURE 18. Comparison of strains R87 and R87P1 dry weight ( © ) , at 37 C, 3 hr, and percentage intracellular water ( O ) , on cellophane-grown cultures, 27 C. Spore types diagrammed correspond to stages of spore development in Figure 17. Percentage intracellular water loss was calculated as maximum percentage of intracellular water minus percentage of intracellular water,- .-..A 54 2 3 4 5 6 COLON Y AGE (DAYS) FIGURE 19. Ea r l y induction of s t r a i n R87P1 sporulation by aeration of pregrown colonies. The dark bar withi n the v e r t i c a l shaded bar represents im-mature spores; the top of the shaded bar represents mature macroconidia. T o t a l growth time was ca l c u l a t e d as the sum of days pre-grown plus sporulation time. Air-flow rate: 50 cm3/min, 27 C. 55 T i i ;—i 1 r 14 10 25 40 75 100 4 0 4 0 0 I u n t r e a t e d a i r 1 d r y h u m i d c o n t r o l a i r a i r AIR F L O W R A T E ( C C / M I N ) FIGURE 20. Variable s p o r u l a t i o n response of A-day pregrown s t r a i n R87P1 to d i f f e r e n t a i r - f l o w rates. The dark bar within the v e r t i c a l shaded bar represents spore i n i t i a t i o n ; the top of the shaded bar represents mature macroconidia. Humid or dry a i r aeration was treated as described i n Materials and Methods. The c o n t r o l f l a s k d i d not r e -ceive a d d i t i o n a l aeration. T o t a l growth time was c a l c u l a t e d as A days pregrown plus s p o r u l a t i o n time. 56 aeration and controlled dehydration were important for early spore induction and maturation. To determine i f early spore induction was dependent on continuous aeration, colonies of strain R87P1, pregrown for 4 days, were separately exposed for periods of 1 to 4 days to aeration at 40 cm3/min. Although there were more spores initiated when the aeration period was long, the pleomorphic strain completed sporulation by 8 days total growth, i n a l l cases, indicating that continuous aeration was not necessary for the completion of the sporulation cycle. To investigate the effect of aeration on strain R87P1, dry weight, and percentage of bound water loss, i t was necessary to treat cellophane-grown colonies in the apparatus reported i n Fig. 16B. To prevent too rapid dehydra-tion of the colony and the medium, air flow was reduced to 10 cm3/min, resulting in delayed sporulation i n i t i a t i o n (8 days) and spore maturation (10 days) (Fig. 20 and 21). The aeration-induced lag phase of 4 days prior to spore i n i t i a t i o n , rapid growth, spore maturation, and 25% bound water loss (Fig. 21) paralleled the spore i n i t i a t i o n cycle of strain R87 (Fig. 18). This similarity suggested that spore production in both strains was equivalent, but the i n i t i a t i o n step was lacking in strain R87P1. Optimum cultural conditions for sporulation. As one of the most obvious visible effects of additional aeration was the dehydration of the culture medium, i t was important to ascertain the optimum cultural conditions for sporulation of both strains, and to determine whether the concentration of the culture medium was responsible for the observed pleomorphic strain sporulation induction. As shown i n Fig. 22, concentration of the medium nutrients permitted mycelial growth and repressed sporulation in both strains. Wild-type M. gypseum sporulated best at low concentrations of glucose-neopeptone. The pleomorphic strain displayed a narrower optimal nutrient concentration range for sporulation than strain R87, 57 FIGURE 21. Comparison of the dry weight and appearance of macroconidia i n aerated ( O ) and nonaerated ( © ) s t r a i n R87P1 colo n i e s . The arrow i n d i c a t e s the beginning of aeration. Spore types diagrammed correspond to the stages of spore development i n Figure 17. Cultures were grown i n cellophane d i s c s and aerated at 10 cm3/min, 27 C. 58 FIGURE 22. Optimum nutrient (glucose plus neopeptone) concentration for strain R87 and strain R87P1 sporulation. Spore development was determined at 3 days ( © ) , 4 days ( O ) , 10 days ( • ) , 12 days ( A ) , 14 days ( A ) , and 16 days (^7). The dotted line represents percentage of spor-ulation of strain R87 after 10 days and strain R87P1 after 16 days, where percentage of spor-ulation was calculated as colony surface area sporulating divided by total colony surface area times 100%. The spore types diagrammed correspond to the stages of spore development in Figure 17. 59 exhibiting a definite requirement for exogenous nutrients and a period of vegetative growth prior to sporulation. Both strains displayed optimum sporulation at 1% glucose-neopeptone concentration in the medium. As shown in Fig. 23, when liquid-grown strain R87 was shifted down from complete medium to agar medium devoid of nutrients, i t was capable of sporul-ation throughout i t s growth cycle, exhibiting maximal sporulation ability prior to the rapid growth phase. The mycelia removed from the submerged culture were completely free of conidia prior to shift-down, indicating that spore i n i t i a t i o n and maturation could be completed in the absence of exogenous nutrients. Strain R87 macroconidia, when placed on the shift-down medium, germinated, formed limited submerged hyphae, formed several aerial hyphae which initi a t e d secondary macroconidial formation after 1 day, and completed sporulation after 3 days. The secondary conidia thus formed also were capable of germination and sporulated after 6 days on the agar surface, but the tertiary macroconidia formed in this cycle were capable only of germination and limited vegetative growth on minimal medium. These results suggested that the mature macroconidium contained suf-ficient endogenous reserves to complete germination, outgrowth and sporulation, and that extensive vegetative growth prior to sporulation was not a prerequisite in the wild strain. The pleomorphic strain, however, was capable only of limited sporulation over a narrow range in the stationary phase of growth, and after a definite period of vegetative growth during which exogenous nutrients were re-quired (Fig. 23). These results paralleled the sporulation cycles for strains R87 and R87P1 reported in Fig. 18, and further suggested that the early sporul-ation of aerated strain R87P1 prior to the rapid growth phase (Fig. 21) was a result of true i n i t i a t i o n of processes which were not normally expressed in that growth phase. Growth of either strain on 1% glucose-neopeptone medium so l i d i f i e d with 60 • i i i i i i i i 1 1 1 1 1 i STRAIN R87P1 SHIFT-DOWN SPORULATION TIME 0 2 4 6 8 10 12 14 S U B M E R G E D ' CULTURE GROWTH (DAYS ) FIGURE 23. Formation of macroconidia after shift-down of strain R87 or strain R87P1 from complete medium to minimal agar medium. The number of days required for mature spore formation is shown as circled numerals associated with the growth curves of strain R87 ( © ) , or strain R87P1 <0). Incubation of mature strain R87 macro-conidia on the shift-down medium is represented by 0 days submerged culture growth, and ( 0 ) represents no spores formed after 10 days incubation after s h i f t -down, 27 C. 61 1.8 to 10% agar (w/v) did not enhance growth or sporulation i n i t i a t i o n , and restriction of nutrient diffusion or fungal exoenzyme diffusion by growing the colony on cellophane or dialysis membranes delayed sporulation by 2 days in both strains. Thus, physical, conditions (i.e., aeration) which resulted in an i n -creased gel concentration in the culture medium and caused decreased nutrient or exoenzyme diffusion appeared to delay sporulation. When either strain was grown on media containing various amounts of concen-trated culture f l u i d by-products, neither growth nor sporulation was stimulated. Strain R87P1 was not stimulated or inhibited i n sporulation a b i l i t i e s by either i t s own culture f l u i d by-products or the by-products of strain R87. Another approach to this consideration was to grow strain R87P1 on cellophane discs (which were perforated to allow hyphal penetration of the medium). Aeration of the colony was carried out to induce sporulation and then the sporulating aerial hyphae were stripped off, so that only the submerged hyphae remained. When the new emergent aerial hyphae were examined for spores, no spores were seen after 10 days despite the fact that the previous aerial hyphae had been sporulating and the culture was now a total of 20 days old. These results suggested that an inducer of sporulation was not present i n the wild-type strain medium or concentrated in the pleomorphic strain medium by aeration. In addition to medium dehydration, another effect of aeration could be the removal of volatile substances, C O 2 , and other gaseous "staling factors" possibly causing the early i n i t i a t i o n of sporulation. When a flask (from Fig. 17A) of strain R87P1 was connected to a flask of strain R87 so that the volatile materials from strain R87P1 would pass over the strain R87 colony, no delay in wild strain sporulation was observed. Also, the accumulation of possible sporulation-inhibitory volatile materials produced by pleomorphic cultures was disproved by the observed sporulation of strain R87P1 cultures at 12 to 14 days (Fig. 18), as this would be the time when any volatile substances would be most concentrated. 62 When s t r a i n R87 was grown i n the presence of added concentrations of 5, 10, and 15% C 0 2 , growth and sporulation were completely i n h i b i t e d . The i n h i b i t i o n was reversed by removing the cultures from the CO2 atmosphere. Growth of the pleomorphic s t r a i n was not affected by the increased CO2 tensions, but sporula-t i o n did not occur i n the presence of 5 to 15% CO2 additions. Nature of the pleomorphic s t r a i n reversion. The pleomorphic spores formed by aerated colonies were of c h a r a c t e r i s t i c wild-type morphology (Fig. 17). Un-l i k e s t r a i n R87 conidia, s t r a i n R87P1 conidia would not germinate i n p h y s i o l o g i c a l s a l i n e (37 C), but d i d form d i s c r e t e germ tubes i n nutri e n t media (37 C). The conidia were f e r t i l e and formed t y p i c a l pleomorphic s t r a i n colonies which sporu-l a t e d very sparsely i n 12 to 14 days without a d d i t i o n a l aeration as did the parent pleomorphic colony. No revertants to s t r a i n R87 morphology, pigmentation, or sporulation c h a r a c t e r i s t i c s were observed. To determine i f the i n i t i a t i o n of s t r a i n R87P1 sporulation by aeration was unique to t h i s p a r t i c u l a r pleomorphic s t r a i n , or was a c h a r a c t e r i s t i c shared by pleomorphic s t r a i n s of other genera and species, numerous independently i s o l a t e d pleomorphic s t r a i n s were aerated as described for s t r a i n R87P1. The r e s u l t s of t h i s screening suggest that the M. gypseum pleomorphic s t r a i n used here was not unique i n i t s response to aeration, and that sporulation i n d i f f e r e n t dermato-phyte genera and species may be i n i t i a t e d i n a s i m i l a r manner (Table 10). The morphology and manner i n which the induced pleomorphic conidia arose from the vegetative hyphae were c h a r a c t e r i s t i c of the respective wild-type s t r a i n . The induced macroconidia were f e r t i l e and germinated to form pleomorphic colonies which were i n d i s t i n g u i s h a b l e , one from the other. TABLE 10. Comparison of different pleomorphic species* response to aeration Appearance of mature macroconidia No. of ~ Pleomorphic strain strains Aerated culture Control culture 8 tested Days Total days Total days aeration b growth growth Microsporum gypseum (R87P1) 1 4 8 14 Microsporum gypseum (R87P12) 1 4 8 14 Microsporum gypseum (R87P8) 1 5 9 No spores c Microsporum gypseum (R87P14) 1 5 9 No spores 0 Microsporum cookei 1 5 10 16 Microsporum canis 1 7 13 19 Microsporum fulvum 1 4 10 20 d Epidermophyton fJoccosura 3 7 13 16 Trichophyton violaceum 1 2 d 15 20 d a No aeration. c A f t e r 20 days growth. b Air flow rate at 40 cm3/min, 27 C. d Microconidia induced, no macroconidia. 64 Discussion: Pleomorphism has been described previously as a degeneration, due to the apparent loss of wild-type functions and c h a r a c t e r i s t i c s (Reiss & Leonard, 1957). Induction of sporulation i n pleomorphic s t r a i n s by aeration, however, suggests that pleomorphism was not a complete sporulation degeneration, as only the a b i l i t y to i n i t i a t e wild-type functions within the normal time sequence was l o s t , not the r e a l a b i l i t y to perform these sporulation functions. A l t e r a t i o n i n n u t r i e n t agar or concentration of growth by products did not appear to be responsible for e a r l y spore induction i n the pleomorphic s t r a i n , nor did the removal of v o l a t i l e " s t a l i n g f a c t o r s " appear to stimulate sporulation. Although carbon dioxide has been found to stimulate sporulation i n microconidiate Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton megnini cultures (Chin & Knight, 1957; Balabanoff, 1963), t h i s gas was found to i n h i b i t sporulation i n both s t r a i n R87P1 and R87 of M. gypseum. Carbon dioxide, s i m i l -a r l y , has been found to i n h i b i t the growth and sporulation of an asporogenous T. mentagrophytes pleomorphic s t r a i n (Chin & Knight, 1957) and to i n h i b i t f r u i t -body formation i n basidiomycetes (Plunkett, 1956; Niederprum & Wessels, 1969). The most s t r i k i n g s i m i l a r i t y concomitant with the appearance of mature spores i n both s t r a i n R87 and R87P1 was the per cent of i n t r a c e l l u l a r water l o s s . Water loss also has been reported to be associated with spore induction and maturation i n slime molds, agari c s , and i n polypores (Plunkett, 1956; Bonner & Shaw, 1957). The primary e f f e c t of aeration, however, was possibly an a l t e r a t i o n i n c e l l sur-face properties, with i n t r a c e l l u l a r water loss being a secondary r e s u l t . Spore formation i n M. gypseum occurred only on a e r i a l hyphae. I t has been suggested by Morton (1961) that the emergence of submerged hyphae into a e r i a l conditions t r i g g e r s a c e l l surface change, due to the creation of an a i r to water i n t e r f a c e , which i n turn acts as a sporulation stimulus. Accumulation 65 of melanin i n the conidia of As p e r g i l l u s nidulans also has been explained by the creation of an a e r i a l a i r to water i n t e r f a c e ( O l i v e r , 1972). I t was possible that the pleomorphic hyphae were unable to undergo the normal wild-type s t r a i n c e l l surface change on emergence i n t o a e r i a l conditions, and hence did not i n i t i a t e sporulation. This lack of response to a e r i a l conditions perhaps was r e f l e c t e d by the i n t r a c e l l u l a r water loss r e s u l t s reported, which show that the a e r i a l hyphae of s t r a i n R87P1 required exposure to a e r i a l conditions for twice as long as the wild-type s t r a i n to lose the same percentage of i n t r a c e l l u l a r water. Additional aeration of the pleomorphic s t r a i n resulted i n more rapid water l o s s , probably through a l t e r a t i o n of the a i r to water equilibrium at the c e l l surface. When sporulating s t r a i n R87P1 hyphae were removed from the agar surface or when pleomorphic spores were germinated, the new emergent aerial, hyphae were t y p i c a l l y asporogenous and pleomorphic. This indicated that the e f f e c t s of aeration were not transmitted to the submerged vegetative hyphae and that the aeration e f f e c t was s t r i c t l y phenotypic and r e v e r s i b l e . Recently i t has been shown that hyphal branching and elongation were c h a r a c t e r i s t i c of a given environment, and that growth a c t i v i t y was expressed at the hyphal t i p (Grove & Bracker, 1970; Katz, Goldstein, & Rosenburger, 1972). Cessation of a p i c a l t i p elongation and branching, without r e s t r i c t i n g nuclear d i v i s i o n , c e l l enlargement, and septation, could describe the processes giving r i s e to macroconidia i n M. gypseum and other dermatophytes. Tatum et a l have shown that p l e i o t r o p i c e f f e c t s leading to a l t e r e d gross morphology were genetic-a l l y determined i n Neurospora, and may represent a l t e r a t i o n of s i n g l e enzymes regulating c e l l w all and membrane ( l i p i d ) synthesis (Brody & Tatum, 1967; Scott & Tatum, 1970). Thus, the pleomorphic s t r a i n possibly arose from a s i m i l a r p l e i o t r o p i c mutation, r e s u l t i n g i n an i n a b i l i t y to balance vegetative hyphal elongation and spore development, possibly owing to an a l t e r a t i o n of enzyme 66 pathways involved i n c e l l wall or membrane synthesis. The primary locus of t h i s mutation could be a defect i n a s i n g l e regulatory enzyme, as genetic studies of M. gypseum have suggested that the pleomorphic a l t e r a t i o n was the r e s u l t of a s i n g l e gene a l t e r a t i o n (Weitzman, 1964). Each of the steps involved i n spore formation ( a p i c a l t i p growth cessation, c e l l expansion, and septation) require the directed, coordinated action of various c e l l organelles. I t has been shown recently that the processes of cytoplasmic streaming and organelle d i s t r i b u t i o n are directed by cytoplasmic filaments (Wessels et a l , 1971) which presumably act as cytoplasmic "pumps". Coordination of the action of these filaments also was c o n t r o l l e d g e n e t i c a l l y . F i b r i l o r i e n t a t i o n directed hyphal growth: i n A s p e r g i l l u s nidulans unipolar d i r e c t i o n resulted i n v e s i c l e formation and multipolar d i r e c t i o n resulted i n metulae formation ( O l i v e r , 1972). Chemical d i s o r i e n t a t i o n of the filaments re s u l t e d i n a l t e r e d hyphal t i p growth (Richmond & Pring, 1971). I t i s pos-s i b l e that these cytoplasmic f i b r i l s d i r e c t hyphal growth by: (a) t r i a l and e r r o r , where only the successful growth form would survive, or (b) external stimulus d i r e c t i o n , where environmentally induced c e l l surface changes r e l a t e changes i n the external medium to the f i b r i l s . From the r e s u l t s of t h i s sec-t i o n , and considering the s p e c i f i c i t y and energy requirements of d i f f e r e n t i a -t i o n , i t i s most l i k e l y that the l a t t e r hypothesis was correct. The pleomor-phic s t r a i n , therefore, possibly arose through i n t e r n a l modifications which a l t e r e d the " s e n s i t i v i t y " of the c e l l u l a r machinery ( f i b r i l s ) to the external stimulus, or as mentioned previously, through a l t e r a t i o n s i n the c e l l w all or membrane which prevented the c e l l from "detecting" changes i n the external environment. The r e s u l t s of t h i s study suggested that the pleomorphic a l t e r a t i o n may be a valuable s t r a i n for the e l u c i d a t i o n of the i n t r a c e l l u l a r and c e l l surface events c o n t r o l l i n g and coordinating hyphal elongation and sporulation. The 67 r e s u l t s also suggested that the "mutation" giving r i s e to pleomorphism may be s i m i l a r i n d i f f e r e n t dermatophyte genera. S i m i l a r i t y i n the i n i t i a t i o n of Microsporum sp. and Epidermophyton floccosum macroconidia and Trichophyton  violaceum microconidia was suggested also. Aeration of asporogenous pleo-morphic c l i n i c a l dermatophyte specimens to a s s i s t i n mycologic diagnosis may also prove to be a valuable technique. 68 Section IV I s o l a t i o n and c h a r a c t e r i z a t i o n of Microsporum gypseum spore coat and mycelial w a l l glycoproteins: involvement i n spore germination Introduction: I t has been proposed previously that the Microsporum gypseum a l k a l i n e germination protease was responsible, at l e a s t p a r t i a l l y , f o r macroconidium germination i n i t i a t i o n (Sections I & I I ) . Ideal l y to confirm t h i s hypothesis, a germination protease s p e c i f i c substrate which contributed to the s t r u c t u r a l i n t e g r i t y of the spore coat should be extracted from the spore coat and employed as an i n v i t r o protease substrate. Fungal spore coat proteins have been reported to be present as d i s c r e t e surface layers (Fisher & Richmond, 1970), as enzymes associated with the spore coat (Bartnicki-Garcia, 1968), as e a s i l y removed proteins i n the form of d i s -crete i n t e r n a l protein l a y e r s , and as glycoproteins (Hunsley & Burnett, 1970). As glycoproteins account for a large proportion of the fungal c e l l w all (Korne & Northcote, 1960) and are important linkages cleaved i n b a c t e r i a l spore germ-i n a t i o n (Warth & Strominger, 1971; Warth, 1972), the r o l e of these polymers i n M. gypseum conidia germination was examined. When one considers the complex s t r u c t u r a l nature of the fungal c e l l wall or spore coat (Bartnicki-Garcia, 1968; Hunsley & Burnett, 1970; Fletcher, 1971; Necas, 1971) i t seems u n l i k e l y that a s i n g l e enzyme could be responsible for s u f f i c i e n t spore coat weakening to allow germ tube emergence. More l i k e l y , t h i s process i s a coordinated event, with several l y t i c enzymes playing a c t i v e r o l e s . These hydrolases may be l o c a l i z e d within the mature spore coat i n l y s o -somal v e s i c l e s (Section I I ) . Just as the coordinated action of d i s u l f i d e reductase and c h i t i n synthetase r e s u l t i n gross morphological changes i n the budding yeast (Nickerson & Falcone, 1954; Cabib & Bowers, 1971.) the coordinated action of spore coat l y t i c enzymes may allow germ tube emergence, without causing total c e l l wall hydrolysis. In an attempt to understand spore germination, a model for M. gypseum macroconidium construction has been proposed, and the observed l y t i c germin ation activities were related to this model. Germination appeared to be a result of the coordinated action of several hydrolases, with the alkaline germination protease possibly acting as the germination-initiating enzyme. 70 Materials and Methods: Culture growth and harvesting. Growth i n l i q u i d cultures has been described previously. Sporulating cultures were grown on agar medium composed of glucose 1% (w/v), neopeptone 1% (w/v) ( D i f c o ) , agar 2% contained i n Roux f l a s k s . At s p e c i f i e d time i n t e r v a l s the a e r i a l sporulating hyphae were scraped o f f the agar surface with a bent glass rod. Spores were p u r i f i e d as described previously (Section I ) . C e l l - f r e e extract and c e l l w all preparation. C e l l - f r e e extracts (CFX) were prepared by breaking mycelia or spores by the modified l i q u i d nitrogen method of Bleyman and Woese (1969). C e l l debris was removed by c e n t r i f u g a t i o n at 10,000 * g f o r 10 min at 4 C. Extracts f o r enzyme determinations were buffered i n 0.05 M sodium b a r b i t a l (Veronal) buffer, pH 8.0. P e l l e t s from CFX preparation were broken further by repeated freeze-thawings and grinding i n a mortar with a p e s t l e i n the presence of l i q u i d nitrogen. Fine glass beads (100 u, Sigma Chemical Co., St. Louis, Mo.) were added to spore preparations to enhance breakage. These were removed l a t e r by repeated s e t t l i n g and decanting the c e l l wall m a t e r i a l . Breakage was estimated as complete when the spores and mycelia appeared free of phase-bright cytoplasmic material, and when the o r i g i n a l structures had been fragmented into very short u n i t s . The resultant p e l l e t was washed eight times i n p h y s i o l o g i c a l s a l i n e (pH 6.5) and twice i n d i s t i l l e d water by vortexing and resedimenting at 600 * g for 2 min. The f i n a l p e l l e t was resuspended i n 10 volumes of 10% sodium dodecyl s u l f a t e (SDS) and extracted i n a shaking water bath (New Brunswick S c i e n t i f i c Co., New Brunswick, N.J.) at 37 C f o r 3 days. The SDS was changed once during t h i s time period. The extracted p e l l e t was washed an additional four times i n d i s t i l l e d water to remove the SDS. The p u r i f i e d spore coats or mycelial walls were drie d i n vacuo over calcium chloride to a constant weight at 25 C. 71 Glycoprotein extraction. Samples for glycoprotein extraction f i r s t were defatted by the method of Barnicki-Garcia et a l (1962), using acid:ethanol:ether (equal volumes 12N HCl to 1:1 95% ethanol + ether). This treatment released most of the pigment from the preparations and gave a white to grey coloured f i n a l product. Glycoproteins were extracted by the method of Korne and Northcote (1960). Routinely, each 100 mg of spore or mycelial material was extracted with 50 ml of ethylene-diamine for 3 days at 37 C in a shaking water bath. The material was sedimented at 600xg for 5 min, and the pellet was washed with ethylene diamine, then methanol and dried in vacuo over calcium chloride, at 25 C. The pooled ethylene diamine washings were concentrated by flash evaporation (30 C) to approximately 5 ml, then precipitated by dropwise addition to 200 ml of methanol. The resultant precipitate was resuspended in 10 ml of water and dialysed against water for 12 hrs at 4 C. The contents of the dialysis sac were centrifuged at 10,000 x g for 10 min at 4 C. The sediment was washed with water, repelleted an4 lyophylized. (water-insoluble Fraction B). The aqueous supernatant and washings were pooled and lyophilized (water-soluble Fraction A). Lyophylization was found to be superior to in vacuo desiccation for dehydration of the f i n a l products, as the latter method produced a sample which was more d i f f i c u l t to dissolve for subsequent analysis. Gel electrophoresis and staining. Electrophoresis was conducted using 5% polyacrylamide gels polymerized as suggested for the Canalco (Rockville, Md.) Model 6 system. The gel and electrophoresis buffer was 0.05 M sodium borate containing 8 M urea, pH 9.5, as recommended by Korne and Northcote (1960). The gel columns measured 0.5 * 6.0 cm and were run at 3 mA per gel, for 90 min at 25 C. Samples were suspended in 10 M urea prior to electrophoresis. Glycoprotein 72 Fraction B was rendered more soluble by heating i n a b o i l i n g water bath f o r 5 to 7 min. D i s s o l u t i o n of F r a c t i o n B was enhanced by the addition of ethylene diamine to the sample at a r a t i o of 0.5:1 (v/v) with 10 M urea. Ethylene diamine also was added to the electrophoresis buffer at a 0.5:1 (v/v) r a t i o for electrophoresis of F r a c t i o n B. As addition of ethylene diamine to the gel mixture prevented or delayed polymerization, gels without ethylene diamine were run at 3 mA per gel for 15 min p r i o r to sample a p p l i c a t i o n . Carbohydrate was detected i n the gels by a modified p e r i o d i c a c i d S c h i f f s t a i n (PAS). The best PAS s t a i n i n g was obtained when 100 yg anthrone-positive material (Morris, 1948) was applied per g e l . A f t e r electrophoresis, gels were fi x e d i n 5% t r i c h l o r o a c e t i c a c i d (TCA) for 2 min, then oxidized i n 7% a c e t i c a c i d : 70% ethanol: 1% periodate (w/v) f o r 1 hr. ( C u l l i n g , 1957; Dulaney & Touser, 1970). The gel was washed i n water to remove the excess periodate and then stained with S c h i f f reagent for 10 min. The S c h i f f reagent was prepared by the combined methods recommended by the Schleicher and Schuell Co. (Keene, New Hampshire) and C u l l i n g (1957), whereby 2 g basic fuchsin was dissolved i n 400 ml b o i l i n g d i s t i l l e d water, then cooled to 50 C, and 10 ml of 2 N HCl was added. The dye then was cooled to 25 C and 4 g sodium metabisulfite was added. This mixture was l e f t f o r 18 hr at 4 C, then was c l a r i f i e d with activated char-coal and f i l t r a t i o n . The c l e a r dye was stable for several months i f stored i n the dark at 4 C. The dyed gels were decolorized with 0.5% sodium metabisulfite f o r one to three days (metabisulfite was changed and prepared fresh d a i l y ) . P rotein was detected by s t a i n i n g with 0.25% (w/v) Coomassie blue i n 7% (w/v) a c e t i c a c i d for 15 min. (Dulaney & Towser, 1970). Destaining was conducted for one to three days i n 7% a c e t i c a c i d . Best r e s o l u t i o n was obtained when 25 yg of protein was applied per g e l . Gels were scanned using a G i l f o r d Model 2410 l i n e a r transport f i t t e d to a 73 Model 2400 recording spectrophotometer. PAS stained gels were scanned at 540 nm and Coomassie blue stained gels were scanned at 560 nm. A transport rate of 2 cm/min and an 0.1 by 2.36 cm aperture plate were employed to maintain band r e s o l u t i o n . Band m o b i l i t i e s were compared by c a l c u l a t i n g the r e l a t i v e m o b i l i t y of each band as distance moved * 60 min electrophoresis time, under standard conditions. Glycoprotein and c e l l w a l l a n a l y s i s . Glycoprotein hexose content was estimated by the anthrone reaction (Morris, 1948) and protein content was estimated by the method of Lowry et a l (1951). The protein content of the ethylene diamine-insoluble residue was estimated a f t e r protein s o l u b i l i z a t i o n with IN NaOH f o r 45 min at 100 C. Glycoproteins (1 mg) f o r amino a c i d analysis were hydrolysed i n vacuo i n 1 ml of 6 N HCl at 108 C for 24 hrs. A f t e r h y d r o l y s i s , the HCl was removed i n vacuo over KOH and concentrated H 2 S O 4 . Amino acids were analysed by the pro-cedure of Spackman, Moore, and Stein (1958) i n a Beckman Model 120 C automatic amino acid analyser. Amino sugars and basic amino acids were determined using an 18 cm column eluted at pH 5.25 (Cameron, 1972). The ester content of the spore coats and mycel i a l walls was estimated by the method of Hesdrin (1949) using B-d-glucose pentaacetate as the standard. D i s u l f i d e s were estimated by the combined methods of Kanetsuna et a l (1972) and Ellman (1959) a f t e r reduction of the c e l l w a l l d i s u l f i d e s with sodium borohydride. The resultant s u l f h y d r y l s were estimated spectrophotometrically with 5,5'-d i t h i o b i s (2-nitro-benzoic acid) (DTNB) reagent. Free s u l f h y d r y l s of the c e l l w a l l preparations were estimated with DTNB without previous reduction with sodium borohydride. Acid-soluble orthophosphate was estimated by the method of Chen et a l (1956) a f t e r washing samples with IN HCl f o r 15 min at 100 C. Acid-insoluble phosphate was estimated a f t e r hydrolysis i n 5N H 2S0 H and 74 concentrated HNO3 in a Kjeldahl apparatus (Fiske & Subbarow, 1925). Titration of the spore coat surface charges was conducted after the method of M i l l (1966). Whole spores were suspended at 106/ml i n d i s t i l l e d water and adjusted to pH 10 with NaOH. Dropwise titration of 0.1 N HCl with constant s t i r r i n g was monitored with a Model 10 pH meter. (Corning Scientific Instruments, Corning, N.Y.). Uniform 0.025 ml drops were formed in a Leur Taper Dropping Tip pipette (Winley-Morris Co. Ltd., Montreal, Canada). Surface phosphates were removed by treating with IN HCl for 15 min at 100 C in a water bath. The spores then were pelleted at 600 x g (2 min) and washed twice with physiological saline (pH 6.5) before titr a t i o n . Spore coats were defatted by washing with chloroform methanol (2:1 v/v) at 37 C, 15 min (Fisher & Richmond, 1969), and then washed as pre-viously mentioned before tit r a t i o n . Spores also were treated with alkaline phosphatase to remove surface phosphates by the method of Fisher & Richmond (1969). Melanin release by germinating spores was estimated as absorbance at 540 nm after diluting the germination supernatant f l u i d samples with an equal volume of IN NaOH. (Bull, 1970). Enzyme determinations. Alkaline protease and alkaline phosphodiesterase activities were determined as previously mentioned (Section II). Esterase activity was estimated using benzoyl-L-tyrosine ethyl ester (BTEE) as substrate and by following absorbance at 256 nm (Hummel, 1959). A modification of the procedure of Kanetsuna et a l (1972) was used to determine 61,3 glucanase activity. Laminarin substrate was suspended at 2mg/ml in 0.05 M sodium acetate buffer, pH 5.0. A reaction mixture composed of 3 ml substrate, 0.1 ml CFX, and 0.4 ml buffer was incubated for 0, 3 and 4 hr at 37 C. The reaction was stopped by adjusting to pH 7 at 4C, and the glucose liberated was estimated by the "glucostat" glucose oxidase reaction (Worthington Biochemical Corp., Freehold, N.J.). Specific activity was determined as the 75 average rate of glucose (ug) released/min/ml per mg CFX protein. This method was s p e c i f i c for g-D-glucose and provided greater s e n s i t i v i t y than the method previously reported (Kanetsuna et a l , 1972). Acetyl glucosaminidase (chitinase) a c t i v i t y was estimated by the method of Loomis (1969) using p-nitrophenol-N-acetyl -8-D-glucosamine as substrate. 76 Results: C e l l w a l l f r a c t i o n a t i o n . As shown i n Figure 24, the germination protease was found to hydrolyse spore coats better than wild type or pleomorphic hyphal c e l l w a l l s , suggesting that p r o t e a s e - s p e c i f i c substrate linkages were present within the spore coats. At l e a s t h a l f of the protein present i n the spore coat or c e l l wall was extractable with ethylene diamine (Table 11). The other portion of the protein (residual protein) i n the preparations remained i n the ethylene diamine i n s o l u b l e residue. The sum of the glycoprotein-protein plus the r e s i d -ual p r otein was i n good agreement with previously reported t o t a l protein values. The ethylene diamine soluble glycoproteins were divided into water-soluble (Fraction A) and water-insoluble (Fraction B) glycoproteins. Fractions A and B electrophoresed as a s i n g l e band and moved i n a l i n e a r fashion a f t e r e l e c t r o -phoresis f o r 60-150 min at 3 mA/gel. The s i n g l e e l e c t r o p h o r e t i c band showed congruence of the PAS s t a i n i n g and Coomassie blue s t a i n i n g bands (Figure 25). Glycoprotein composition fluctuations with sporulation a c t i v i t y . As shown i n Table 12, Fraction A remained r e l a t i v e l y constant as percent dry weight of mycelial walls and spore coats. Fraction B, however, was at l e a s t twice as concentrated i n the spore coats as compared with the mycelial walls. As would be predicted from Table 12, Fraction B increased i n concentration/dry weight as sporulation progressed. Analysis of the r a t i o of glycoprotein-protein to glycoprotein-hexose material i n the glycoproteins extracted at d i f f e r e n t time i n t e r v a l s showed that the r a t i o of protein:hexose also increased i n Fraction B during early sporulation, and then decreased with spore maturation (Figure 26). The r a t i o of protein:hexose i n 7 day spores was 3.8:1 as compared to 2.0:1 i n asporogenous mycelia. Although the protein:hexose r a t i o for Fraction A was found to increase and decrease during sporulation, a s i m i l a r f l u c t u a t i o n was observed i n the asporogenous mycelial F r a c t i o n A. When 7 day spores were 77 FIGURE 24. Comparison of c e l l walls and spore coats as a l k a l i n e protease substrates. Enzyme a c t i v i t y was measured as ug substrate protein released/min/mg enzyme protein. Substrates used were germinated s t r a i n R87 spore coats ( • ) , s t r a i n R87 5 day mycelia ( O ) , and asporogenous pleomorphic s t r a i n (R87P1) 5 day mycelia ( • ) . 78 TABLE 11. D i s t r i b u t i o n of protein i n i s o l a t e d mycelial walls and spore coats % dry weight Sample Total Residual Total Previous t o t a l glycoprotein- p r o t e i n 3 protein protein value* 3 protein S t r a i n R87 mycelia 5.2 6.0 11.2 10.6 S t r a i n R87 5 day sp o r e s c 6.39 4.77 11.16 -S t r a i n R87 7 day spores 8.14 5.28 13.42 12.9 a ethylene diamine i n s o l u b l e residue b Leighton & Stock, 1970a c corrected for mycelial protein contribution. 79 FIGURE 25. Congruent electrophoresis of PAS and Coomassie blue staining bands from glycoprotein preparations. A. Fraction A, strain R87 5 day-old mycelia; B. Fraction B, strain R87 5 day-old mycelia; C. Fraction A, strain R87 spores; D. Fraction B, strain R87 spores. PROT. = Coomassie blue staining band (protein). CHO = PAS staining band (carbohydrate). TABLE 12. Percent of unfractionated c e l l wall dry weight that i s ethylene diamine extractable glycoprotein % of unfractionated c e l l w all dry weight A a B b C c pleomorphic mycelia 2.5 3.6 82.5 R87 mycelia 2.9 4.7 85.7 B17 mycelia 2.3 5.7 74.3 R87 spores 3.5 10.0 70.5 a water-soluble glycoprotein, Fraction A k water-insoluble glycoprotein, Fraction B c ethylene diamine i n s o l u b l e residue. FIGURE 26. Changes i n glycoprotein p r o t e i n : hexose r a t i o s during s p o r u l a t i o n . A. s t r a i n R87, F r a c t i o n A glycoprotein (O) and F r a c t i o n B glycoprotein ( @) B. asporogenous pleomorphic s t r a i n , F r a c t i o n A glycoprotein (O) and Frac t i o n B glycoprotein (@) 82 germinated, the protein:hexose r a t i o remained r e l a t i v e l y constant i n Fraction A, but decreased dramatically i n Fraction B (Figure 26). Ele c t r o p h o r e t i c m o b i l i t i e s on borate-urea gels suggested differences between the i n d i v i d u a l Fraction A & B glycoproteins. Electrophoresis of Fraction A revealed i d e n t i c a l m o b i l i t i e s f o r spore coat and mycelial wall preparations (Figure 27). Electrophoresis of Fraction B, however, revealed d i f f e r e n t m o b i l i t i e s , with the spore coat f r a c t i o n having the greatest m o b i l i t y . (Figure 28). Comparison of glycoprotein amino a c i d compositions. As shown i n Table 13, the amino a c i d composition of the Fraction A glycoprotein from both s t r a i n R87 spores and pleomorphic mycelia were quite s i m i l a r , d i f f e r i n g by 3% i n pr o l i n e residues and les s than 3% i n the other residues. Fraction A was notably r i c h i n p r o l i n e > threonine > glycine > glutamic acid > s e r i n e . F r a c t i o n A was low , i n tyrosine > i s o l e u c i n e > phenylalanine > arginine > methionine > h a l f - c y s t i n e . Comparison of the Fract i o n A glycoprotein amino a c i d residues r e l a t i v e to l y s i n e also showed the s i m i l a r i t y i n these f r a c t i o n s , despite t h e i r d i f f e r e n t o r i g i n s (Table 14). By t h i s method of comparison the differences i n the residue r a t i o of each amino a c i d was less than or approximately 1.0. Fraction A appeared r i c h i n p r o l i n e > threonine > glycine > glutamic a c i d > serine, i n t h i s comparison, j u s t as i t did i n Table 13. The low r a t i o amino acids also appeared i n the same order as those i n Table 13. When Fracti o n A glycoproteins from e i t h e r source were hydrolysed and chromatographed on the 18 cm column for basic amino acids, the resultant t r a c i n g showed four ninhydrin-positive a r t i f a c t peaks. (Figure 29). These peaks were not obtained when Fract i o n B from e i t h e r s t r a i n R87 spores or pleomorphic hyphae was hydrolysed and chromatographed under the same conditions. Fraction B appeared quite d i s s i m i l a r from Fraction A, being r i c h i n leucine, rather than p r o l i n e (Table 13). Fraction B from s t r a i n R87 spores appeared Electrophoretic mobilities of glycoprotein Fractions A (Coomassie blue stain) on 5% polyacrylanide gels electrophoresed in the presence of 0.05 M Borate buffer and 8 M urea, pH 9.5. A. strain R87 mycelia (5 day); B. pleomorphic strain mycelia (5 day); C. strain R87 ungerminated spores. 84 i 1 1 1 1 r 1.6 i 1 — - \ 1 1 ^ 1 B i i i I I i 0 1 2 3 4 5 — - M I G R A T I O N ( c m ) — • FIGURE 28. Different electrophoretic mobilities of glycoprotein Fractions B (Coomassie blue stain) on 5% polyacrylamide gels electrophoresed in the presence of ethylene diamine (see Methods). A. strain R87 mycelia (5 day); B. pleo-morphic strain mycelia (5 day); C. strain R87 ungerminated spores. 85 TABLE 13. Comparison of amino acid and amino sugar compositions of glycoproteins from asporogenous hyphae and from macroconidia Percent of t o t a l nmoles amino a c i d Amino Acid Residue Fraction A Fraction B Pleomorphic S t r a i n R87 Pleomorphic S t r a i n R87 mycelia spores mycelia spores Glycine 11.4 Alanine 9.0 Valine 7.8 Isoleucine 0.5 Leucine 2.0 Serine 1.0.7 Threonine 1.3.4 Aspar t i c Acid 5.6 Glutamic Acid 9.1 Lysine 3.0 H i s t i d i n e 7.2 Arginine 0.2 Phenylalanine 0.3 Tyrosine 0.6 Ha l f - c y s t i n e ND Methionine 0.4 P r o l i n e 16.0 Glucosamine 2.3 Galactosamine 0.4 11.0 7.4 10.2 7.2 4.2 6.5 5.0 2.8 4.5 1.2 1.9 3.1 2.3 23.9 15.6 9.7 4.6 7.2 15.3 4.5 5.2 5.7 4.6 11.0 11.8 4.5 8.5 3.9 9.7 1.2 4.4 1.6 0.3 0.4 . 1.4 0.2 0.9 9.5 7.2 1.0 1.5 2.2 ND ND trace trace 1.6 6.6 19.8 4.7 6.9 0.5 11.6 3.5 trace trace ND Note: ammonia accounted for _< 2.0 nmole % of t o t a l ND = not detectable TABLE 14. Comparison of amino acid and amino sugar composition of glycoproteins r e l a t i v e to l y s i n e content Relative ymole amino a c i d 3 Amino „ „. . „ . ., Fr a c t i o n A Fraction B Acid Residue Pleomorphic S t r a i n R87 Pleomorphic S t r a i n R87 mycelia spores mycelia spores Glycine Alanine Valine Isoleucine Leucine 3.81 3.00 2.59 0.16 0.68 2.80 1.83 1.27 0.31 0.59 0.76 0.44 0.29 0.19 2.45 8.3 5.24 3.68 2.50 12.68 Serine Threonine 3.55 4.46 2.47 3.89 0.47 0.46 5.86 4.26 Aspartic Acid Glutamic Acid 1.87 3.05 1.47 3.00 0.47 0.46 8.89 6.89 Lysine H i s t i d i n e Arginine 1.0 2.42 0.07 1.0 1.13 0.11 1.0 0.17 0.14 1.0 0.20 0.20 Phenylalanine Tyrosine 0.09 0.21 0.22 0.25 0.97 0.16 ,84 ,79 Half - c y s t i n e Methionine ND 0.14 ND trace ND 0.17 trace 5.32 Pr o l i n e 5.35 5.06 0.49 5.58 Glucosamine Galactosamine 0.77 0.13 0.14 trace 1.19 trace 2.87 ND a compared to Lysine (= 1.0) Note: data derived from Table 12 ND = not detectable C H A R T .D IRECTION FIGURE 29. Effluent peaks from the short column for basic amino acids, using the pleomorphic strain Fraction A glycoprotein hydrolysate. Ninhydrin-positive peaks were measured at 570 nm. Artifact peaks are numbered, other peaks are: G l c N H 2 = glucosamine, GalNH2 = galactosamine, Lys = lysine, His = histidine, NH3 = ammonia, AGPA = L-a-amino-B-guanidino-propionic acid (internal standard). 00 88 s i m i l a r to that from the pleomorphic mycelia except that the spore f r a c t i o n contained increased a s p a r t i c a c i d > methionine > glutamic a c i d , greatly decreased l y s i n e > leucine > glucosamine, and almost no h i s t i d i n e and arginine. F r a c t i o n B from the spores also contained the only detectable amounts of h a l f - c y s t i n e , and as mentioned previously, contained increased methionine. The other residues d i f f e r e d with the pleomorphic mycelia F r a c t i o n B residues by * 3%. Comparison of amino a c i d residues r e l a t i v e to l y s i n e indicated great d i f -ferences between the amino a c i d r a t i o s of Fraction B and Fraction A, further i n d i c a t i n g that these f r a c t i o n s were quite d i f f e r e n t (Table 14). S t r a i n R87 spore and pleomorphic mycelia Fraction B glycoproteins also showed great d i f -ferences i n amino a c i d composition by t h i s comparison, undoubtedly because of the low concentration of l y s i n e i n the spore f r a c t i o n (1.2%) and the high con-centration of l y s i n e i n the pleomorphic f r a c t i o n (9.7%). Thus, s t r a i n R87 spore Fraction B had a greater r a t i o of a l l amino a c i d residues, except the basics , than the pleomorphic mycelia. Amino sugars were present i n a l l the glycoprotein f r a c t i o n s examined. The pleomorphic mycelia Fraction A contained the only measurable quantity of galactosamine (Figure 29 & Table 13). The spore f r a c t i o n s consistently con-tained less glucosamine than the mycelial f r a c t i o n s , and the Fraction B glyco-proteins from both sources contained more glucosamine than t h e i r respective Fr a c t i o n A glycoproteins. Glycoprotein f r a c t i o n s as protease substrates. When the germination pro-tease was tested f o r a c t i v i t y against the Fraction A & B glycoproteins, the re s u l t s did not show an absolute preference by the germination protease f or eith e r glycoprotein f r a c t i o n . When amino nitrogen release due to hydrolysis was measured, i t appeared that both Fractions A & B could serve as substrates for the germination protease. Examination of the decreased protein content of 89 F r a c t i o n A & B a f t e r 1 hr hydrolysis showed a s i m i l a r trend. (Table 15). Very l i t t l e h y d r o l y t i c a c t i v i t y was observed when the ethylene diamine i n s o l u b l e r e s i d u a l protein was used as a germination protease substrate and the rate of myce l i a l wall and spore coat r e s i d u a l protein hydrolysis was found to be the same. Glycoprotein composition fluctuations during s t a r v a t i o n . Examination of s t r a i n R87 mycelia during submerged growth and s t a r v a t i o n showed a pattern of glycoprotein proteinrhexose r a t i o s quite opposite to those i n Figure 26. In t h i s case the protein:hexose r a t i o of Fraction A increased during growth and decreased during s t a r v a t i o n from 9 days onward (Figure 30 B). The pro t e i n : hexose r a t i o of Fraction B remained r e l a t i v e l y constant during growth and s t a r v a t i o n . The decrease i n Fraction A protein:hexose r a t i o s t a r t i n g at 9 days was accompanied by an increase i n i n t r a c e l l u l a r a l k a l i n e protease a c t i v i t y (Figure 30 A). Other hydrolases i n germinating spores. I t was apparent from the preceding r e s u l t s that the germination protease may not s p e c i f i c a l l y hydrolyse only one spore coat glycoprotein i n v i t r o . A survey of other hydrolase a c t i v i t i e s was conducted to determine i f other l y t i c enzymes may be involved i n spore germin-a t i o n . As shown i n Table 16, a l k a l i n e protease, BTEE esterase, and c h i t i n a s e were present i n greatest a c t i v i t y i n the spore p e l l e t s . Glucanase was present at highest a c t i v i t y i n the spore CFX and a l k a l i n e phosphodiesterase was concen-tr a t e d i n the mycelial walls. Greatest a c t i v i t y of the a l k a l i n e protease and spore coat glucanase appeared i n ungerminated spores (0 h r ) . Spore coat a l k a l i n e phosphodiesterase, BTEE esterase, and c h i t i n a s e peaked i n a c t i v i t y during germ tube emergence (3 h r ) . A l l p e l l e t a c t i v i t i e s decreased during germ tube elongation (6 h r ) . L o c a l i z a t i o n of spore coat phosphates. Phosphate ester cleavage and TABLE 15. Comparison of Fract i o n A and B glycoproteins as a l k a l i n e protease substrates Glycoprotein uM amino N release/hr Decreased protein a f t e r 1 hr a f t e r 4 hr Mg/min Spores Fraction A 1.92 0.84 1.04 Fr a c t i o n B 1.92 0.86 2.57 Pleomorphic mycelia Fraction A 1.07 1.11 Fracti o n B 1.92 1.18 Wild type mycelia F r a c t i o n A 1.15 0.80 2.56 Fraction B 2.22 0.68 1.38 91 FIGURE 30. Changes i n the glycoprotein protein:hexose r a t i o s with s t a r v a t i o n . A. Increased i n t r a c e l l u l a r a l k a l i n e protease a c t i v i t y with s t a r v a t i o n ( O ) . B. Comparison of the protein:hexose r a t i o s of l i q u i d grown s t r a i n R87 mycelia Fra c t i o n A ( O ) and F r a c t i o n B ( © ) . TABLE 16. Hydrolase a c t i v i t i e s i n mycelial and germinating spore f r a c t i o n s S p e c i f i c A c t i v i t y i n P e l l e t S p e c i f i c A c t i v i t y i n CFX Enzyme My c e l i a 3 0 hr spores 3 hr spores 6 hr spores M y c e l i a3 0 hr spores 3 hr 6 hr spores spores A l k a l i n e Protease 0 4.77 17.65 12.2 10.0 3.0 5.79 2.75 7.6 Al k a l i n e Phosphodies t e r a s e c 33.29 1.53 4.02 1.47 14.5 2.22 1.08 1.96 BTEE Esterase** 0.21 0.16 0.44 0.08 0 0 0 0 6 1:3 Glucanase e 1.2 3.21 1.71 1.51 1.04 4.60 4.50 4.50 C h i t i n a s e f 3.4 37.27 47.23 37.23 1.6 3.78 1.91 4.58 3 5 day old asporogenous pleomorphic hyphae b AOD 280 nm * 10 3 /min/mg protein c AOD 410 nm * 10 3 /min/mg protein d uM substrate used/min/mg protein e ug glucose released/min/mg protein f AOD 420 nm x 10/min/mg protein 93 phosphate release have been Implicated i n spore germination and germination regulation (Sections I & II). Estimation of the phosphate concentration of acid-ethanol washed mycelial wall and spore coat preparations showed that acid-nonextractable phosphate accumulated i n the developing spore coats (Figure 31). Acid-extractable phosphate accounted for 64.0 ug/mg dry weight of spores and 38.2 ug/mg dry weight of mycelia. Total phosphate was calculated as 6.78% dry weight for ungerminated spores and 3.97% dry weight for the mycelia. Titration of ungerminated, germinated, and acid-washed spores suggested that a portion of the acid-labile phosphate groups was present on the surface of the spores. As shown in Figure 32 (a f i r s t differential plot of the t i t r a -tion results), the spores lost acidic surface charges with germination or when ungerminated spores were washed with acid. Washing ungerminated spores with chloroform-methanol did not alter the ungerminated spore t i t r a t i o n curve. Examination of the acid wash solution revealed that considerable orthophosphate was present. A similar shift in tit r a t i o n curve was brought about by treating ungerminated spores with alkaline phosphatase. The ti t r a t i o n curve for 18 hr germinated spores suggested that the surfaces of the germ tubes and developing hyphae were quite different from that of the ungerminated spores. Examination of other c e l l wall linkage groups. Examination of other c e l l wall linkage groups gave results consistent with those in Table 17. Ester cleavage occurred during i n i t i a l germ tube emergence (3 hr). The acid-soluble and acid-insoluble phosphate groups decreased in concentration after i n i t i a l germination. Disulfide cleavage did not appear to occur during spore germin-ation and, as would be expected, free sulfhydryl groups also remained constant during this period. The ester and acid-insoluble phosphate content of mycelial walls was less than that of the spores (Table 17). These results may correspond to the greater mycelial ac t i v i t i e s of the l y t i c enzymes which cleave the preceding 94 3 4 5 DAYS GROWTH FIGURE 31. Incorporation of acid-insoluble phosphate into spore coats during sporulation. Liquid-grown strain R37 hyphae (GJ) and liquid-grown strain R87P1 hyphae (®) were used to determine the mycelial wall acid-insoluble phosphate content. Surface cultures of sporulating strain R87 were used to determine the acid-insoluble phosphate content of developing spore coats (O). 95 FIGURE 32. F i r s t d i f f e r e n t i a l p l o t o f the r e s u l t s o f t i t r a t i o n o f t h e s u r f a c e groups p r e s e n t on M. gypseum s t r a i n R87 m a c r o c o n i d i a w i t h 0.1 N H C l . The c o n t r o l t i t r a t i o n w i t h o u t a d d i t i o n a l s p o r e s appears i n the f i r s t peak ( @ ) . Ungerminated s p o r e s had m o s t l y a c i d i c s u r f a c e groups (O), which were l o s t a f t e r a c i d washing (A) o r a f t e r 4 h r g e r m i n a t i o n ( A ) . Spores germinated f o r 3 8 h r (•) p o s s e s s e d more a l k a l i n e s u r f a c e p r o p e r t i e s t h a n ungerminated o r 4 h r ge r m i n a t e d s p o r e s . 96 TABLE 17. C e l l wall and spore coat linkage content Linkage Germinating spores 5 day o l d Pleomorphic Mycelia Group 0 hr 3 hr 6 hr Carbohydrate-protein e s t e r 3 0.15 0.05 0.13 0.32 D i s u l f i d e b r i d g e b 0.41 0.42 0.81 0.091 Free s u l f h y d r y l b 0.025 0.035 0.034 0.066 Acid- i n s o l u b l e Phosphate 0 3.8 4.0 2.2 1.5 Acid-soluble Phosphate 0 64.0 54.9 28.1 38.2 3 umoles ester / mg dry weight b 10_t* M / mg dry weight c yg / mg dry weight 97 bonds (Table 16). Free s u l f h y d r y l groups were present i n higher concentration and d i s u l f i d e s i n lower concentration i n the mycelia, possibly i n d i c a t i n g more d i s u l f i d e cleavage i n the mycelia. Acid-soluble phosphate also accumulated i n the mycelial w a l l s , but not to the same concentration as ungerminated spores, and apparently was not present as a surface layer (Figure 32). As shown i n Figure 33, the spore germination supernatant f l u i d became more pigmented as germination progressed,at l e a s t p a r t i a l l y due to the release of melanin. Pigment was released immediately upon addition of the spores to the germination medium. Pigment release was most rapid from 0 to 2 hr and then was l i n e a r to 10 hr. FIGURE 33. Pigment release from germinating s t r a i n R87 macroconidia. 99 Discussion: Glycoproteins have been reported to be present i n Neurospora crassa c e l l walls as thick f i b r i l s which are l a i d down subapically and possibly provide e l a s t i c strength to the hyphae (Hunsley & Burnett), 1970). In nu t r i e n t ex-haustion, glycoprotein and glucan f i b r i l s extend over the hyphal apex, causing d e l i m i t a t i o n of hyphal growth (Hunsley & Burnett, 1970; Robertson, 1959). As previously shown (Section I I I ) , sporulation i n M. gypseum appears to r e s u l t from cessation of a e r i a l a p i c a l t i p elongation, with b r i e f l y - c o n t i n u e d c e l l ex-pansion and septation. This observation suggested that c e l l w all components necessary for the cessation of hyphal elongation were also important i n M. gypseum sporulation. During a p i c a l growth i n Mucor r o u x i i , Phytophthora p a r a c i t i c a , Neurospora  crassa and Schizophyllum commune, short c h i t i n polymers are inserted at the a p i c a l t i p and are further polymerized subapically (Bartnicki-Garcia & Lippman, 1969; Gooday, 1971). Chitinase has been reported to be involved with conidium morphogenesis i n Dictyostelium discoideum and Microsporum gypseum (Loorais, 1969; Leighton & Stock, 1970b). R i g i d i f i c a t i o n of the conidium wall may be rela t e d to melanization (Fletcher 1971; O l i v e r , 1972). Tyrosinase and melanin synthesis have been shown to be important i n M. gypseum sporulation (Leighton & Stock, 1970b) and melanin production was shown to be induced by growth l i m i t a t i o n (Rowley & P i t t , 1972). Melanin may bind to the c e l l wall proteins, creating the very stable "tanned" protein linkage observed i n melanized tissues (Mercer, 1961), thus contributing to the spore coat r i g i d i t y . Melanin and i t s precursors a l s o have been reported to i n h i b i t c h i t i n a s e and glucanase a c t i v i t i e s (Kuo & Alexander, 1967). Thus, concentration of melanin or i t s precursors i n a p i c a l t i p s may i n i t i a t e a p i c a l d e l i m i t a t i o n , by i n h i b i t i n g these depolymerase enzymes and allowing c h i t i n and glucan polymers to extend over the hyphal t i p . Glucan 100 synthesis probably follows chitin polymerization closely, as the two polymers appear to be structurally complementary and united (Hunsley & Burnett, 1970; Wang & Bartnicki-Garcia, 1970). The results i n Figure 26 suggested that the water-insoluble glycoprotein proteinrhexose ratio increased specifically during sporulation. After 5 days sporulation, there was a decrease in this ratio, possibly due to increased hexose (glucan) synthesis. Despite this decrease in proteinrhexose ratio during spore maturation, the total glycoprotein-protein content of the spores was greater than either the immature spores or the mycelia (Table 11). The decrease in water-insoluble glycoprotein-protein during spore germination impli-cated this spore coat fraction as the germination protease-specific substrate. The amino acid analysis of the glycoprotein fractions revealed that the Fraction A glycoproteins from both strain R87 spores and pleomorphic (Strain R87P1) mycelia were of similar composition, suggesting that Fraction A had undergone very l i t t l e compositional transformation during sporulation. Proline accounted for 16 to 20% of the total amino acid content of this water-soluble glycoprotein. This glycoprotein fraction also was high in basic, hydroxy-containing, and acidic amino acids, and low in aromatic and sulfur-containing amino acids. The presence of considerable quantities of aspartic acid/glutamic acid, threonine/serine and lysine/histidine suggested that this fraction was i n -volved with bonding between glycoprotein and chitin and/or glycoprotein and glucan (Sentandreu & Northcote, 1968; Wang & Bartnicki-Garcia, 1970). The presence of measurable quantities of galactosamine i n this fraction may impli-cate this glycoprotein in the observed immunological properties of Microsporum  sp. and other dermatophytes (Grappel et a l , 1969). The artifact peaks, observed in Figure 29, also have been detected by paper chromatography (Applegarth & Bozoian, 1967) and by column chromatography (Cameron, 101 1972). The a r t i f a c t s were believed to r e s u l t from the hydrolysis of gluco-samine i n the presence of amino acids. These a r t i f a c t u a l peaks were not found when glycoprotein Fraction B was hydrolysed, despite the increased glucosamine content i n t h i s f r a c t i o n . As the mode of amino acid linkage to glucan has been reported to influence the rate and r e l a t i v e release of amino acids from c e l l wall hydrolysates (Sentandreu & Northcote, 1968), i t was possible that the amino acid-carbohydrate linkages and subsequent configuration of the Fraction B glycoprotein was s u f f i c i e n t l y d i f f e r e n t from that of the Fraction A glycoprotein to cause a d i f f e r e n t rate or sequence of h y d r o l y s i s , r e s u l t i n g i n no a r t i f a c t production. Glycoprotein F r a c t i o n B from spores was shown to be d i f f e r e n t from that of the mycelia a f t e r both a t o t a l and r e l a t i v e amino a c i d content comparison (Table 13 & 14). The Fraction B glycoprotein was notably r i c h i n leucine, which accounted for 16 to 24% of the t o t a l amino acid composition. This amino ac i d , i n combination with the other a l i p h a t i c amino acids may contribute to the w a t e r - i n s o l u b i l i t y of t h i s f r a c t i o n , which could r e s u l t from exposed hydrophobic side-chains or from intermolecular apolar bonding. Fraction B from-'spores was notably low i n basic amino acids, which resulted i n an increased r a t i o for a l l other amino acids, as compared to the mycelial F r a c t i o n B (Table 1.4). This compositional a l t e r a t i o n could give the spore glycoprotein Fraction B an i n -creased a c i d i c charge, owing to the increased predominance of a s p a r t i c and glutamic acids, possibly accounting for the increased e l e c t r o p h o r e t i c mobility of t h i s f r a c t i o n observed i n Figure 28. The spore glycoprotein Fraction B contained a much larger quantity of methionine than any of the other f r a c t i o n s . Methionine has been reported to increase the r i g i d i t y of the yeast c e l l w a l l , causing dimorphism of Candida  albicans (Mardon et a l , 1969; Mardon & B a l i s h , 1971). Methionine may play 102 a similar role in the construction of the Fraction B glycoprotein, possibly increasing the rig i d i t y of the spore coat. The failure to detect half-cystine in the glycoproteins may be significant, as cysteine and cystine have been reported in a discussion by Roy and Landau (1972) to be absent from many fungal c e l l walls. As these residues are destroyed by acid hydrolysis, or may be obscured by artifacts upon amino acid analysis (Cameron, 1972), independent analysis of these residues should be conducted before their presence is discounted (Raftery & Cole, 1963). Although intact spore coats served as a good protease substrate in vitro (Figure 24), the use of spore coat glycoproteins as in vitro protease substrates did not give clear cut results. The glycoproteins in situ most probably have a very specific conformation in association with other spore coat constituents. Ethylene diamine extracted glycoproteins by mild alkaline hydrolysis of the ester links between the glycoprotein-protein and c e l l wall glucans (Nickerson, 1963), which undoubtedly results in some structural changes in the glycoprotein after the release of these constraints. Furthermore, the Fraction B glyco-protein was insoluble in the assay system, whereas in the spore coat the germ-ination protease was inserted within the meshes of i t s substrate (Section II). Thus, immediately after spore rehydration, M. gypseum spore germination may be initiated by alkaline protease cleavage of spore coat glycoproteins. This conclusion was based on the observations that: (1) germination was prevented by phenyl methyl sulfonyl fluoride, a germination protease inhibitor (Leighton & Stock, 1970a), (2) alkaline protease activity increased specifically on sporulation (Figure 9), (3) alkaline protease was the only l y t i c enzyme which had greater spore coat activity than c e l l wall or CFX activity (Table 16), (4) alkaline protease release was a very early event (Table 16, Figure 2), (5) alkaline protease was inhibited i n vivo after germination i n i t i a t i o n 103 (Section I ) , (6) the a l k a l i n e protease substrate may be the only uniquely d i f f e r e n t spore coat component not found i n the mycelial c e l l w a l l . The water-insoluble glycoprotein was probably the main s i t e of protease cleavage, with increased e l a s t i c i t y of the spore coat r e s u l t i n g . I n i t i a t i o n of germination also was accompanied by increased ?> 1,3 glucanase a c t i v i t y . This enzyme may be required to s t r i p glucan o f f c h i t i n polymers to expose the l a t t e r for subsequent c h i t i n hydrolysis (Manocha & C o l v i n , 1967; B u l l , 1970). Another early germination event was the rapid release of melanin, which prob-ably allowed i n t r a - c e l l w a l l glucanase and c h i t i n a s e to be a c t i v e , and maintain the. growing a p i c a l t i p s t a t e . Later germination events were continued proteolysis and a burst of c h i t i n a s e a c t i v i t y . BTEE esterase a c t i v i t y probably re s u l t e d i n further degradation, by cleaving ester bonds l i n k i n g glycoprotein-protein and glucans. (Nickerson, 1963). A l k a l i n e phosphodiesterase a c t i v i t y could be responsible for glucan or mannan phosphodiester cleavage r e s u l t i n g i n phosphate release and concomitant protease i n a c t i v a t i o n (Sections I & I I ) . The occurrence of maximum phosphodiesterase a c t i v i t y a f t e r i n i t i a l germination was important, as t h i s would prevent premature protease i n a c t i v a t i o n p r i o r to germination i n i t i a t i o n . The high mycelial l e v e l of phosphodiesterase further suggested that t h i s enzyme may be important i n normal hyphal a p i c a l growth, and hence,germ tube elongation. Because of the regulatory r o l e that phosphate played i n spore germination, i t was desirable to determine where phosphate was located i n the spore coat. Acid-insoluble phosphate accumulated s p e c i f i c a l l y with sporulation and was present i n greater concentration i n spore coats than mycelial walls. The a c i d -i n s o l u b l e f r a c t i o n was considered to be phosphodiesters, owing to t h e i r r e s i s t -ance to acid h y d r o l y s i s . Despite the lower concentration of t h i s phosphate linkage i t s s t r u c t u r a l contribution would s t i l l be greater than that of 104 orthophosphate, with phosphodiesters possibly contributing to the polymeric structure of mannans or mannan-protein complexes. (Grappel et a l , 1969; Thieme & Ballou, 1972). As shown in Table 16, there was more acid-soluble than acid-insoluble phosphate in the spore coat. As the spore coats had been defatted, i t was unlikely that this phosphate was present as phospholipid. Ash values pre-viously calculated for M. gypseum (undefatted) accounted for 8% of the spore coat and 11% of the mycelial wall (Leighton & Stock, 1970a). The total phosphate values calculated here accounted for 6.78% of the spore coat and 3.97% of the mycelial wall. These results suggest that phospholipid was probably a minor constituent in M. gypseum spore coats, but may have been more prominent in the mycelial wall. This hypothesis is supported by the recent observation of Dill et al (1972), that lipid was a major constituent of the macromolecules synthesized during germ tube outgrowth and (mycelial) elong-ation by this organism. Spore titrations suggested that acid-soluble phosphate groups, initiall y present on the ungerminated spore external surface, were lost during germin-ation. Conidia of Penicillium expansum similarly have been shown to have surface phosphate groups (Fisher & Richmond, 1.969). Acid-soluble inorganic polyphosphate also has been reported to be on the surface of Neurospora mycelia (Harold, 1962). Binding of phosphate in both these cases was through surface amino sugar groups. The increased basicity of M. gypseum spores after germin-ation or acid washing also was suggestive of an underlying layer of amino groups. The outer electron-dense layer of Microsporum gypseum conidia, ob-served in Figures 12 & 13, may have been due to melanin concentration (Oliver, 1972) or i t may have been due to surface phosphate, pyrophosphate or polyphos-phate binding of the uranium ions of the uranyl. nitrate post-stain (Rothstein 105 & Meier, 1951). I t also should be noted that both polyphosphate and phospho-die s t e r s could serve as diesterase substrates, as both have the appearance of phosphodiesters. Although the d i s u l f i d e concentration of spore coats was greater than the mycelial walls, d i s u l f i d e cleavage did not appear to occur during spore germ-i n a t i o n . The lower d i s u l f i d e and higher s u l f h y d r y l content of the mycelial walls, however, may confirm the observation that d i s u l f i d e cleavage was important i n hyphal growth and branching (Robson & Stockley, 1962). The a l k a l i n e protease also appeared to function i n the turnover of c e l l w a l l glycoproteins during s t a r v a t i o n . Except for hyphal branching and anastomoses, the fungal hyphal wall usually i s regarded as a fixed structure which does not undergo further modification once polymerized. However, turnover of P e n i c i l l i u m and A s p e r g i l l u s c e l l w a l l glucan has been shown a f t e r extreme starvation (Corina & Munday, 1971; T r i n c i & Righelato, 1970). Simi-l a r l y , the b a c t e r i a l c e l l wall was thought to be a stable structure, but extensive turnover of B a c i l l u s s u b t i l i s mucopeptide and techoic acid during logarithmic growth has been shown by Mauck and Glasar (1970). Considering the ease of extraction and non-involvement with sporulation, i t was u n l i k e l y that the water-soluble glycoprotein contributed much to the actual r i g i d i t y of the spore coat, although i t may f i l l the meshes between s t r u c t u r a l glucan, c h i t i n , and water-insoluble glycoprotein. This hypothesis was supported by the observation that turnover of P e n i c i l l i u m fungal wall con-s t i t u e n t s did not cause a decrease i n wall thickness, but did decrease w a l l density ( T r i n c i & Righelato, 1970). The observed increase i n the p r o t e i n : hexose r a t i o of the water-soluble glycoprotein from 5 to 9 days growth may represent early hexose turnover, with protein turnover occurring l a t e r i n s t a r v a t i o n . This r e s u l t was e n t i r e l y l o g i c a l , as hexose would serve as a 106 higher energy y i e l d i n g endogenous substrate than would prot e i n . Hydrolysis of the glycoprotein-protein was probably accomplished by massive e x t r a c e l l u l a r r e -lease of a l k a l i n e protease, following disruption of the lysosomal and v e s i c u l a r hydrolase systems within the starving c e l l s . The electron micrographs of T r i n c i and Righelato (1970) support t h i s hypothesis, as they showed that starving Pencillium hyphae were f u l l of disoriented membranes and v e s i c l e s l a t e i n s t a r v a t i o n . Turnover of fungal c e l l wall glycoprotein-protein probably represented a terminal stage of s t a r v a t i o n , where the c e l l was no longer v i a b l e or reviveable. The released a u t o l y s i s products, however, were probably useful to adjacent hyphae for maintenance metabolism. 107 Section V A l t e r a t i o n of the Microsporum gypseum germination protease p h y s i c a l and p h y s i o l o g i c a l properties by inorganic phosphate: enhanced k e r a t i n o l y t i c a c t i v i t y Introduction: The k e r a t i n o l y t i c a c t i v i t y of dermatophytes has been a subject of i n t e r e s t f o r several years (Stahl et a l . 1949; Raubitscheck, 1961; Chesters & Mathison, 1963). Native k e r a t i n o f f e r s a formidable b a r r i e r to enzymatic attack, due to the complementary structure of the component keratins, the high d i s u l f i d e con-tent, and the i n t r a c e l l u l a r and i n t r a f i b r i l l a r cement which binds the e n t i r e structure together (Frazer et a l , 1959; Mercer, 1951). P u r i f i e d keratinase i s r e l a t i v e l y i n e f f e c t i v e alone i n digesting native k e r a t i n (Yu, Harmon, & Blank, 1969). Mechanical penetration by the mycelial "eroding complex" undoubtedly enhances k e r a t i n digestion (English, 1962). Chemical reduction of k e r a t i n d i s u l f i d e s p r i o r to general k e r a t i n o l y s i s also has been described f o r the clothes-moth digestion of wool (Powning & Irzykiewicz, 1960), for Streptomyces fradiae k e r a t i n o l y s i s (Noval & Nickerson, 1959) and recently f or Microsporum gypseum digestion of human h a i r (Kunert, 1972). The dermatophyte, M. gypseum i s transmitted from host to host by spores. When these spores germinate, an a l k a l i n e protease i s the only protease released during germination and outgrowth (Leighton & Stock, 1970a). The num-ber of spores which w i l l be i n i t i a t e d to germinate i s con t r o l l e d by phosphate released from the germinating spores (Sections I & II) which i n h i b i t s the a l k a l i n e germination protease. This i n h i b i t i o n i s not complete, however, as at l e a s t 50% of the enzyme's o r i g i n a l a c t i v i t y remains a f t e r germination has been completed (Section I ) . 108 The i n v e s t i g a t i o n described here was conducted to determine i f the germ-i n a t i o n protease of M. gypseum also functioned as a keratinase and to deter-mine i f phosphate mediated changes i n the enzyme's phys i c a l and k i n e t i c c h a r a c t e r i s t i c s . As native k e r a t i n appears to be reduced by s u l f i t o l y s i s p r i o r to p r o t e o l y t i c attack by the keratinase complex, a l k a l i n e t h i o g l y c o l -l a t e extracts of native keratins (Jones & Mecham, 1943) were used as enzyme substrates. Also, chemical agents which may denature or be denaturation products of native k e r a t i n i n vivo, were examined to determine i f these agents could a l t e r the a c t i v i t y or structure of the germination protease and keratinase. A model for sequential protease and keratinase action i n the digestion of native k e r a t i n , based on the r e s u l t s of t h i s section, i s presented. Evidence for a common o r i g i n and de r i v a t i o n of the germination protease and keratinase i s also described. 109 Materials & Methods: Keratinase induction. Methods of growth and CFX preparation have been described previously (Sections I & I I ) . Keratinase was induced by the method of Yu, Harmon and Blank (1968) using a medium composed of 0.09% (w/v) glucose and 0.06% (w/v) magnesium s u l f a t e (,7H20) i n 0.028 M phosphate b u f f e r pH 7.8. Hair (guinea p i g , human or horse) or chicken feather, cut i n t o 0.5 cm lengths, was washed thoroughly with 0.1% (w/v) sodium docecyl s u l f a t e to remove surface contaminants, then with 1:1 (v/v) methanol and water with shaking for 18 hrs, 27 C. The k e r a t i n was f i l t e r e d through Miracloth (Calbiochem., La J o l l a , C a l i f . ) to remove the solvents and the f i n a l k e r a t i n residue was washed with d i s t i l l e d water, then 95% ethanol and a i r - d r i e d . The washed h a i r or feather (0.5 gm) was added to 100 ml of medium i n a 250 ml Erlenmeyer f l a s k and was rou t i n e l y a u t o c l a v e - s t e r i l i z e d for 15 min at 121 C. The medium was inoculated with 5 day-old liquid-grown mycelia, which had been sheared by V i r - T i s homogen-atio n (Model 23, V i r - T i s Inc., Gardiner, N.Y.) for 30 sec at approximately 16,000 rev/min. A f t e r i n o c u l a t i o n , the medium was allowed to stand for 5 days and then was shaken for 7 days at 27 C on a B u r r e l l w r i s t - a c t i o n shaker ( B u r r e l l Corp., Pittsburgh, Penn.). Estimation of k e r a t i n percent u t i l i z a t i o n . Keratin u t i l i z a t i o n was estimated by the method of Chesters and Mathison (1963). F i r s t , the t o t a l weight of h a i r s covered with mycelium was estimated by f i l t r a t i o n and desiccation i n vacuo over calcium chloride. Then the dry weight of the mycelium was estim-ated a f t e r d i s s o l u t i o n of the h a i r by treatment with 50 ml of 10% (w/v) NaOH at 96 C f o r 10 min. The value thus obtained for the weight of mycelium a f t e r e x t r a c t i o n was corrected for mycelium weight loss due to NaOH extraction by multi p l y i n g by the c o e f f i c i e n t 1.60. This value was obtained by t r e a t i n g M. gypseum s t r a i n R87 liquid-grown hyphae i n a s i m i l a r manner with NaOH and 110 c a l c u l a t i n g the hyphal weight l o s s . The weight of h a i r remaining a f t e r hyphal growth was, therefore, calculated as the weight of (hair + mycelium) - (weight of mycelium), and the percent u t i l i z a t i o n of keratin was cal c u l a t e d as the ( o r i g i n a l weight of h a i r - weight of h a i r remaining) * o r i g i n a l weight of h a i r x 100%. A l l estimations were done i n t r i p l i c a t e . Keratin extraction and f r a c t i o n a t i o n . Natural k e r a t i n sources (hair or feather) were fract i o n a t e d by a method u t i l i z i n g a l k a l i n e t h i o g l y c o l l a t e extrac-t i o n (Jones & Mecham, 1943) and the f r a c t i o n a t i o n scheme of C o r f i e l d et a l (1958) (Table 18). The 8 k e r a t i n and whole h a i r f r a c t i o n s (1 mg dry weight/ml) were resuspended i n 0.05 M phosphate buffer, pH 9.0 for routine enzyme assays. The prot e i n concentration of the a k e r a t i n was estimated by the method of Lowry et a l (1951) and then was resuspended at 0.5 mg protein/ml of 0.05 M phosphate buffer, pH 7.0. Enzyme assay procedures. Guinea p i g h a i r r o u t i n e l y was used as a source of a and B k e r a t i n or casein substrate. The mixture was incubated at 37 C for 60 min. The reaction was stopped by the addition of 1.0 ml of 10% (w/v) per-c h l o r i c a c i d (PCA) and the resultant p r e c i p i t a t e was removed by ce n t r i f u g a t i o n at 1000 x g, 2 min, 27 C. Blanks contained the complete reaction mixture, but were a c i d i f i e d with PCA p r i o r to incubation. Studies in v o l v i n g the e f f e c t s of metal ions, cations, anions, or urea on enzymic a c t i v i t y were preincubated with the enzyme f or 30 min at 37 C. One unit of a c t i v i t y was equal to an o p t i c a l density change of 0.001 at 280 nra/ml of enzyme/min. S p e c i f i c a c t i v i t y was calculated as units/mg enzyme prot e i n . The determination of pH p r o f i l e s was made by assays using e i t h e r 0.05 M Veronal or 0.05 M phosphate buffers adjusted with HCl or NaOH for the pH 5.5 to pH 9.0 range. A c t i v i t i e s at pH 9.0 to pH 10.0 also were assayed using 0.05 M Borax-NaOH or 0.05 M Borax-NaOH-phosphate buffers. I l l TABLE 18. Keratin Extraction Procedure 20 gm h a i r or 40 gm feathers i n 1000 ml 0.1 N NH30H + 0.5 M sodium t h i o g l y c o l l a t e f 40 C, 24 hr (feather) or 48 hr (hair) extracted 2X f i l t e r through 1 layer Miracloth residue unextracted h a i r & $ k e r a t i n p a r t i c u l a t e f i l t r a t e cone, a c e t i c acid added dropwise to pH 4 centrifuge at 8,000 x g, 25 min, 4 C sediment - dissolve i n 500 ml 0.1 N N H 3 O H , 12 hr, 25 C - centrifuge at 8,000 x g, 15 min, 4 C sediment - wash with 0.1 N N H 3 O H - wash with 95% ethanol - dry i n vacuo over calcium chloride 6 k e r a t i n sediment resuspend i n a minimal volume of 0.1 N N H 3 O H supernatant discard supernatant - add this s o l u t i o n dropwise to 1000 ml (95%) ethanol containing 2.2 ml concen-trated a c e t i c acid. - adjust to pH 4.0 with cone. a c e t i c acid - centrifuge at 8,000 x g, 25 min, 4 C supernatant - discard a k e r a t i n 112 Esterase a c t i v i t y was measured using benzoyl-L-tyrosine e t h y l ester (BTEE) as substrate (Hummel, 1959). The i n h i b i t i n g e f f e c t s of inorganic phosphate and phenyl methyl s u l f o n y l f l u o r i d e (PMSF) were followed spectrophotometrically with a G i l f o r d Model 2400 recording spectrophotometer ( G i l f o r d Instrument Laboratories, Inc., Oberlin, 0.). Sephadex chromatography. Columns (45 cm x 1.5 cm) containing e i t h e r Sephadex G100 or G200 gels were used ro u t i n e l y i n these studies. E i t h e r 0.05 M phosphate b u f f e r pH 7.8 or 0.05 M Veronal buffer pH 9.0 was used for gel rehydra-t i o n and e l u t i o n . The columns were c a l i b r a t e d by the method recommended for Sephadex gels ("Gel f i l t r a t i o n i n theory and p r a c t i c e " , a booklet from Pharmacia, Uppsala, Sweden) using c r y s t a l l i n e ribonuclease A, chrymotrypsin, ovalbumin and aldolase. Enzyme samples applied to the columns contained 0.3 to 0.4 o p t i c a l density 280 nm units/ml. The column drop-former was constructed from a 50 y l disposable pipette,-and the flow rate was 1 drop/10 sec. (or 1 ml/6 min). Fractions of 85 drops or approximately 2.5 ml were c o l l e c t e d on a Warner-Chilcot Model 1205-E2 f r a c t i o n c o l l e c t o r (Canal I n d u s t r i a l Corp., R o c k v i l l e , Md.). Forty f r a c t i o n s were c o l l e c t e d before a p p l i c a t i o n of a new sample. Protein concentration i n each f r a c t i o n was estimated as the o p t i c a l density at 280 nm. A l l columns were run at 25 C. Enzyme concentration. A l k a l i n e protease and keratinase were concentrated e i t h e r by d i a l y s i s against 20% (w/v) polyethylene g l y c o l (PEG) or 20% (w/v) F i c o l l (Pharmacia Fine Chemicals, Uppsala, Sweden) i n 0.05 phosphate or Veronal b u f f e r s . Other methods of concentration were f l a s h evaporation at 25 C and diaflow d i a l y s i s concentration i n an Amicon Model 52 U l t r a f i l t r a t i o n c e l l (Amicon Corp., Lexington, Mass.). D i a l y s i s concentration i n the presence of reducing agents was conducted a f t e r adding 45 mM cysteine or 5 mM d i t h i o t h r e i t o l (DTT) to the 20% PEG s o l u t i o n (Cleland, 1964). 113 Other conditions. A pleomorphic s t r a i n (R87P1) of M. gypseum. asporogenous under normal c u l t u r a l conditions, was induced to sporulate by increased aeration methods (Section I I I ) . Cultures were grown i n 250 ml Erlenmeyer flasks con-t a i n i n g 100 ml of agar medium composed of 1% (w/v) glucose, 1% (w/v) Neopeptone (Dico), 18% (w/v) agar and d i s t i l l e d water (pH 6.5). Sporulation of 4 day pre-grown cultures was i n i t i a t e d a f t e r 2 days of aeration at 40 cm3/min and mature spores were formed a f t e r 4 days t o t a l aeration. Control cultures without a d d i t i o n a l aeration and aerated cultures were harvested d a i l y by scraping the a e r i a l hyphae from the agar surface with a bent glass rod. Three flasks were harvested at each time period to give s u f f i c i e n t material for further studies. 114 Results: Phosphate stimulation of keratinase a c t i v i t y . As shown i n Figure 2, inorganic phosphate caused a 50% decrease i n e x t r a c e l l u l a r germination protease a c t i v i t y a f t e r approximately 8 hr germination. The germination protease, how-ever, was not completely i n h i b i t e d , even a f t e r 12 hr germination. When the germination protease from 10 hr germinated spore supernatant was assayed for a c t i v i t y at d i f f e r e n t pH l e v e l s , a peak of a c t i v i t y at pH 7 was detected, which was not present i n 3 hr germinated spore supernatants (Figure 34). When the 10 hr germination protease was assayed for a c t i v i t y i n the presence of phosphate buf f e r , the protease a c t i v i t y at pH 9 decreased and the a c t i v i t y at pH 7 showed a marked increase (Figure 34). As shown i n Table 19, this s h i f t i n pH optimum was accompanied by a change i n s p e c i f i c i t y f o r substrate, when compared with the i n t r a c e l l u l a r a l k a l i n e protease. Further examination of the apparent increased preference for k e r a t i n substrates showed that the 10 hr germination protease hydrolysed whole k e r a t i n and 3 k e r a t i n , and that a c t i v i t y against a ke r a t i n was increased by phosphate ions (Table 20). Figure 35A shows how inorganic phosphate dramatically de-creased c a s e i n o l y t i c a c t i v i t y at pH 9, and at the same time increased a keratino-l y t i c a c t i v i t y at pH 7.0. As shown i n Figure 35B, phosphate also decreased the BTEE esterase a c t i v i t y of the 3 hr germination protease. Neither the 10 hr germination protease nor a keratinase preparation possessed detectable BTEE esterase a c t i v i t y . Figures 36 and 37 show the pH a c t i v i t y p r o f i l e s of the 10 hr germination protease and the e x t r a c e l l u l a r M. gypseum keratinase using a and 3 keratins as substrate. The germination protease d i d not display exceptional a c t i v i t y p r i o r to phosphate addition. A f t e r phosphate addition, however, the germination protease showed enhanced a c t i v i t y towards both k e r a t i n sources, and displayed 315 l 1 ; 1 1 r 6 7 8 9 10 pH FIGURE 34. Alteration in the germination protease pH optimum on casein in the presence of i n -organic phosphate. Germination protease activity was measured after 3 hr germination ( O ) and after 10 hr germination (©) in 0.05 M Veronal buffer. Protease collected at 10 hr also was assayed in the presence of 0.05 M Phosphate buffer ( A ) . Specific activity = AOD 280 nm x 103/min/mg enzyme protein. 116 TABLE 19. Comparison of i n t r a c e l l u l a r and e x t r a c e l l u l a r a l k a l i n e protease s p e c i f i c i t y f o r substrate Relative S p e c i f i c A c t i v i t y 3 Substrate b I n t r a c e l l u l a r 0 A l k a l i n e Protease E x t r a c e l l u l a r ^ A l k a l i n e Protease Casein 1.0 1.0 Native Albumin 0.03 0.01 Heat Denatured Albumin 0.58 0.82 Bovine Serum Albumin 0.08 0.06 Haemoglobin 0..25 0.20 Ge l a t i n 0.23 0.20 Protamine 0.40 0.20 K e r a t i n e 0.09 0.50 a S p e c i f i c A c t i v i t y = yg protein released/min/mg enzyme protein a l l substrates suspended at 1% (w/v) i n 0.05 M Veronal buf f e r pH 8.0 c MW 33,000 from Sephadex G100 chromatography ^ from 10 hr germinated spores supernatant e whole guinea pig h a i r . 117 TABLE 20. Relative a c t i v i t y of 10 hr germination protease on keratin f r a c t i o n s Enzyme Mixture S p e c i f i c a c t i v i t y 5 unfractionated . . b , . h a keratxn h a i r D k e r a t i n germination protease 1.0 4.8 germination protease + 10" 5 M inorganic phosphate 0.80 3.68 3.32 germination protease + 1 0 - 3 M t h i o s u l f a t e keratinase 1.32 1.0 5.0 8.0 4.12 10.48 a S p e c i f i c a c t i v i t y of protease = AOD 280 nm * 103/min/mg enzyme protein, a l l values r e l a t i v e to germination protease a c t i v i t y on unfractionated h a i r b from guinea p i g h a i r c induced by growing on guinea p i g h a i r I I I I I L__ 0 IO'6 1 0 ' 5 1Cr 4 10-3 io- 2 (M ) ION CONCENTRAT ION FIGURE 35 A shows the i n h i b i t o r y e f f e c t of inorganic phosphate on the 3 hr germination protease c a s e i n o l y t i c a c t i v i t y at pH 9 i n 0.05 M Veronal b u f f e r ( © ) and the stimulatory e f f e c t of inorganic phosphate on the 3 hr germination protease a k e r a t i n o l y t i c a c t i v i t y at pH 7 i n 0.05 M Veronal b u f f e r (O) . FIGURE 35 B shows the i n h i b i t o r y e f f e c t of inorganic phosphate (0) and PMSF (•) on the BTEE esterase a c t i v i t y of the 3 hr germination protease. S p e c i f i c a c t i v i t y of protease = A0D 280 nm * 103/min/mg enzyme pr o t e i n . Esterase s p e c i f i c a c t i v i t y = uM substrate used/min/mg enzyme prot e i n . 119 FIGURE 36. pH a c t i v i t y p r o f i l e s of the 10 hr germination protease i n 0.05 M Veronal buffer ( © ) , 0.05 M Phosphate buffer (O), 0.1 M phosphate bu f f e r (A) , and the e x t r a c e l l u l a r keratinase i n 0.05 M phosphate buffer (•) on a ke r a t i n substrate. S p e c i f i c a c t i v i t y = AOD 280 nm x 103/min/mg enzyme prot e i n . r 120 FIGURE 37. pH a c t i v i t y p r o f i l e s of the 10 hr germination protease i n 0.05 M Veronal buffer (@) and 0.05 M phosphate bu f f e r ( O ) , and the extra-c e l l u l a r keratinase i n 0.05 M Veronal buffer (ffl) and 0.05 M phosphate buffer (•) on 8 k e r a t i n substrate. S p e c i f i c a c t i v i t y = OD 280 nm x 10 3/min/ml enzyme pr o t e i n . 121 sharper pH optima at pH 7 f o r a k e r a t i n and at pH 9 for 6 k e r a t i n . A d d i t i o n a l phosphate concentrations also enhanced the B k e r a t i n o l y t i c a c t i v i t y of the keratinase. Evidence for germination protease subunit structure. As previously mentioned, the molecular weight of the 3 hr germination protease was estimated at 30,000-33,000 by disc gel electrophoresis (Figure 1). This value also was obtained when G100 or G200 Sephadex chromatography was used (0.05 M Veronal b u f f e r , pH 7.8) (Figure 38A). However, when the enzyme was chromatographed i n the presence of phosphate, or was c o l l e c t e d at l a t e r germination times (6-10 h r ) , the bulk of the protein chromatographed at 16,000 molecular weight (MW), and the enzyme a c t i v i t y was dispersed through the column f r a c t i o n s (Figure 38B). Thus, i t was considered that phosphate affected the s t a b i l i t y of the germination protease, possibly s p l i t t i n g the 33,000 MW enzyme in t o 16,000 MW subunits which lacked a l k a l i n e protease a c t i v i t y , but possessed l i m i t e d keratinase a c t i v i t y . The enzyme peaks i n Figure 38A were considered to be aggregates of the d i s -sociated protease, as they a l l ran at MW multiples of 16,000. When the i n t r a c e l l u l a r a l k a l i n e protease from sporulating hyphae was examined by G200 Sephadex chromatography, the 33,000 MW species was found to be the major peak at 5 days, during spore maturation (Table 21), as would be predicted from Figure 9. However, lower MW a l k a l i n e proteases were found i n abundance at 1, 2 and 4 days i n immature spores. An apparent gradient from low MW (8,000) at 1 day (hyphae only) to higher MW (33,000) at 5 days was evident. Very high MW species (120,000) also were obtained at 2, 4 and 5 days. The f i r s t appearance of these higher MW species coincided with the f i r s t appear-ance of the 16,000 MW protease species and with the onset of sporulation at 2 days. 122 M O L E C U L A R WEIGHT >150,000 67000 49,000 33,000 16,000 I 1 1 1 1 1 6 8 10 12 14 16 18 20 FRACT ION N U M B E R FIGURE 38. E l u t i o n of the 3 hr germination protease from a Sephadex G100 column with 0.05 M Veronal buffer pH 9 (A) and e l u t i o n of the 3 hr germination protease from a Sephadex G100 column with 0.05 M phosphate buffer pH 7.8 (B). The s o l i d l i n e repre-sents p r o t e i n / f r a c t i o n . A l k a l i n e protease a c t i v i t y ( © ) and a keratinase a c t i v i t y (O) were estimated i n each f r a c t i o n . 8 keratinase a c t i v i t y was congruent with the a l k a l i n e protease peaks. Units of a c t i v i t y = AOD 280 nm/min/ml f r a c t i o n . 123 TABLE 21. Molecular species of the alkaline protease during sporulation Colony Age Specific a c t i v i t y 3 8,000 MW 16,000 MW 33,000 MW 120,000 MW 1 day 1280 7.4 0 0 2 days 63 920 26.7 475 4 days 46 300 380 300 5 days 16 6.4 920 385 3 Specific activity = AOD 280 nm * 102/min/mg CFX protein. 124 Keratinase molecular weight and aggregation. M. gypseum possesses d e f i n i t e keratinase a c t i v i t y when grown on k e r a t i n substrates as the sole carbon and nitrogen source (Table 22). Mutant s t r a i n B17 and the pleomorphic s t r a i n of M. gypseum which have a l t e r e d germination protease s p e c i f i c i t y (Leighton & Stock, 1970b) also had lower k e r a t i n o l y t i c a c t i v i t y than the wild type s t r a i n R87. When the e x t r a c e l l u l a r keratinase was c o l l e c t e d , concentrated by f l a s k evaporation and chromatographed, most of the protein and a k e r a t i n o l y t i c a c t i v i t y was found to elute at 16,000 MW (although much of the enzyme's a c t i v i t y was l o s t during p a r t i a l p u r i f i c a t i o n ) (Figure 39A). S t r a i n R87 hyphae, harvested at various stages of sporulation, showed that the i n t r a c e l l u l a r keratinase a c t i v i t y peak preceded the i n t r a c e l l u l a r a l k a l i n e protease a c t i v i t y peak (Figure 40A). This r e s u l t was consistent with the data reported i n Table 21, which showed that the 16,000 MW protease species preceded the appearance of the 33,000 MW a l k a l i n e protease species. The pleomorphic s t r a i n was found to be devoid of both a l k a l i n e protease and keratinase a c t i v i t y (Figure 40A & B). Shortly a f t e r forced aeration and i n i t i a t i o n of sporulation i n pleomorphic cultures the appear-ance of both a l k a l i n e protease and keratinase a c t i v i t i e s was induced, but the protease peak preceded the keratinase peak. As the pleomorphic s t r a i n i n i t i a l l y has no a l k a l i n e protease a c t i v i t y , the early protease peak may have represented induction of the 8,000 MW species. This appeared to be the case, as the 16,000 MW keratinase a c t i v i t y peak (Figure 40B) immediately followed the a l k a l i n e prote-ase peak (Figure 40A). Keratinase concentration by d i a l y s i s against polyethylene g l y c o l (PEG), F i c o l l , or by diaflow u l t r a f i l t r a t i o n followed by chromatography resulted i n the i n a c t i v a t i o n pf the 16,000 MW peak and increased e l u t i o n (as compared to Figure 39A) of the enzyme protein at 33,000 MW and 150,000 MW (Figure 39B). TABLE 22. Per cent u t i l i z a t i o n of k e r a t i n sources by s t r a i n s of M. gypseum % u t i l i z a t i o n a f t e r 13 days growth Keratin Source BOT * T,m pleomorphic s t r a i n R87 s t r a i n B17 v ^ / s t r a i n human h a i r 39.3 21.4 9.3 horse h a i r 53.1 38.9 10.9 guinea p i g h a i r 53.1 42.0 8.0 chicken f e a t h e r 3 24.5 13.5 6.3 3 steam s t e r i l i z e d 126 M O L E C U L A R WE IGHT > 150,000 33,000 16,000 I 1 —1 1 4 6 8 10 12 14 16 18 20 22 24 F R A C T I O N N U M B E R i FIGURE 39. E l u t i o n of the e x t r a c e l l u l a r keratinase from Sephadex G100 with 0.05 M Phosphate bu f f e r pH 7.8 a f t e r concentration by f l a s h evaporation (A) or by PEG or F i c o l l d i a l y s i s ( B ) . Enzymic a c t i v i t y was measured i n each f r a c t i o n using casein ( ® ) or a k e r a t i n ( O ) as substrate. The s o l i d l i n e represents protein e l u t i o n from the column. Units of a c t i v i t y = AOD 280 nm * 10 3/mih/ml f r a c t i o n . FIGURE 40. Comparison of the i n t r a c e l l u l a r a l k a l i n e protease a c t i v i t y (A) and the time of appearance of i n t r a c e l l u l a r keratinase a c t i v i t y (B) i n s t r a i n R87 during s p o r u l -at i o n ( , the pleomorphic s t r a i n under normal growth conditions ( © ) , and the pleomorphic s t r a i n under increased aeration ( O ) . S p e c i f i c a c t i v i t y = AOD 280 nm * 103/min/mg CFX p r o t e i n . 128 Aggregation of the enzyme protein also was obtained when i s o l a t e d Sephadex G100 enzyme preparations were rechromatographed on Sephadex G100. When i s o l a t e d 65,000 MW keratinase was rechromatographed on G100, some of the enzyme protein and a c t i v i t y eluted at 16,000 MW but most eluted at > 150,000 MW (Figure 41A) . Very l i t t l e p r o t e i n eluted at 65,000 MW. S i m i l a r l y , when i s o l a t e d keratinase of 33,000 MW was rechromatographed on G100, almost a l l of the enzyme protein and a c t i v i t y eluted at 65,000 MW, rather than 33,000 MW (Figure 41B). Re-chromatography of i s o l a t e d 16,000 MW keratinase also gave an array of peaks, with f r a c t i o n s e l u t i n g at 16,000 MW, 33,000 MW, 65,000 MW, and > 150,000 MW (Figure 41C). These r e s u l t s suggested that i n the process of rechromatography or d i a l y s i s , some fa c t o r necessary f o r the a c t i v i t y and separate existence of the 16,000 MW unit was l o s t . Neither rechromatography nor d i a l y s i s against PEG or F i c o l l of the 16,000 MW germination protease u n i t , however, promoted aggregation. This indicated that although the keratinase and (germination protease + inorganic phosphate) possessed the same molecular weight, some a d d i t i o n a l a l t e r a t i o n had affected the 16,000 MW keratinase species so that i t would aggregate upon d i a l y s i s or rechromatography. Also i t was found that the PEG-concentrated keratinase (> 150,000 MW) increased i n a c t i v i t y with repeated freezing and thawing (Figure 42). Chroma-tography of the keratinase a f t e r 20 days frozen storage revealed that the large MW complex was disaggregated p a r t i a l l y into smaller u n i t s , the sum t o t a l a c t i v i t y of which was greater than the i n t a c t complex alone (Table 23). Stimulation of keratinase a c t i v i t y with autoclaved feather solubles. During the i n v e s t i g a t i o n of keratinase digestion of various natural k e r a t i n sources, i t was found that autoclaved chicken feathers served as an excellent substrate, but steam or e t h a n o l - s t e r i l i z e d feathers were u t i l i z e d poorly (Table 24). 3 29 M O L E C U L A R WEIGHT >150,000 65,000 33,000 16,000 6 8 10 12 14 16 18 2 0 22 FRACT ION NUMBER FIGURE A l . Aggregation of the a l k a l i n e protease and keratinase a f t e r rechromatography of i s o l a t e d Sephadex G100 peaks (phosphate b u f f e r system) of molecular weight 65,000 (A), 33,000 (B) and 16,000 (C) on Sephadex G100 and eluted with 0.05 M phosphate buffer pH 7.8. The s o l i d l i n e represents protein e l u t i o n from the column. A l k a l i n e protease (9) and a keratinase ( O ) a c t i v i t y were calculated as AOD 280 nm * 10 3/ min/ml f r a c t i o n . 130 DAYS STORAGE FIGURE 42. Increase i n the c a s e i n o l y t i c a c t i v i t y of the large molecular weight keratinase (>150,000) a f t e r storage at -10 C and repeated freeze-thawing. S p e c i f i c a c t i v i t y = AOD 280 nm x 10/min/mg enzyme prot e i n . 131 TABLE 23. E f f e c t of storage at -10 C on the molecular weight and enzyme a c t i v i t y of the large molecular weight keratinase Before cold storage Af t e r cold storage 3 A c t i v i t T o t a l Molecular 0 T o t a l b Molecular 0 c I V I y S p e c i f i c b Weight S p e c i f i c Weight A c t i v i t y D i s t r i b u t i o n A c t i v i t y D i s t r i b u t i o n A l k a l i n e Protease a keratinase 6 keratinase 215.5 1 a l l 76.4 >150,000 82.2 633.2 217.8 168.2 >150,000 66,000 >150,000 66,000 33,000 16,000 >150,000 66,000 3 at -10 C f o r 20 days b S p e c i f i c a c t i v i t y = a c t i v i t y u n i t s / f r a c t i o n OD 280 nm Where one a c t i v i t y u n i t = AOD nm x 10 3/min/ml f r a c t i o n and the t o t a l s p e c i f i c a c t i v i t y i s the sum of the i n d i v i d u a l peaks of a c t i v i t y for each enzyme type 0 Molecular weights calculated from Sephadex G100 e l u t i o n p r o f i l e s using 0.05 M phosphate buffer pH 7.8 132 The near complete u t i l i z a t i o n of autoclaved feathers was not e n t i r e l y due to heat denaturation of the native protein structure, as autoclaved and resuspended feather was digested to the same degree as the steam s t e r i l i z e d feather sub-s t r a t e . S i m i l a r l y , i s o l a t e d 6 k e r a t i n was u t i l i z e d j u s t as well as the steamed feather. Isolated a k e r a t i n proved to be a poor growth substrate. The r e s u l t s i n Table 24 suggested that a soluble factor present i n the autoclaved chicken feather supernatant (AFS) was enhancing the u t i l i z a t i o n of the chicken feather substrate. When AFS was added to the i s o l a t e d keratinase, the enzyme's a c t i v i t y was increased. D i l u t i o n of the > 150,000 MW enzyme caused a decrease i n a and 8 keratinase a c t i v i t y and an increase i n a l k a l i n e protease a c t i v i t y (Figure 43A). D i l u t i o n of the same keratinase preparation i n the presence of AFS enhanced the a c t i v i t y of the 8 keratinase and decreased the a c t i v i t y of the a l k a l i n e protease (Figure 43B). When the AFS was s l u r r i e d with a mixed bed of H + and C l Dowex, with H + Dowex, or was dialysed, i t l o s t i t s a b i l i t y to enhance 8 keratinase a c t i v i t y . This r e s u l t d i d not occur, however, when the AFS was s l u r r i e d with C l Dowex or was deproteinized with PCA. This suggested that the factor i n the AFS, which enhanced a and 8 keratinase a c t i v i t y , was a small and negatively-charged non-protein molecule. Stimulation of keratinase a c t i v i t y with reducing agents. As shown i n Figure 44, d i l u t i o n of the keratinase i n the presence of the reducing agents cysteine, s u l f i t e , b i s u l f i t e , t h i o s u l f a t e and d i t h i o t h r e i t o l (DTT) enhanced the a c t i v i t y of the keratinase and the a l k a l i n e protease when compared to Figure 43A. Cysteine, l i k e AFS, was the only anion which s p e c i f i c a l l y en-hanced the a c t i v i t y of the keratinases (Figure 44C). The a c t i v a t i o n by s u l f i t e , b i s u l f i t e , cysteine, or DTT, however, was much greater than that caused by AFS. TABLE 24. E f f e c t of the method of s t e r i l i z a t i o n on the percent u t i l i z a t i o n of chicken feather k e r a t i n by M. gypseum „, % utilization after Treatment , _ , 13 days growth autoclaved 3 96.4 steamed b 24 .5 e t h a n o l 0 9.7 autoclaved, f i l t e r e d and 28.0 resuspended g k e r a t i n f r a c t i o n (autoclaved) a k e r a t i n f r a c t i o n (autoclaved) 31.8 <5 3 feathers suspended i n medium and autoclaved at 121 C, 15 min. D feathers steamed at 121 C, 15 min., then suspended i n s t e r i l e medium c feathers soaked i n 95% ethanol f o r 18 hr, then f i l t e r e d , a i r d r i e d , and resuspended i n s t e r i l e medium FIGURE 43. Comparison of the e f f e c t of d i l u t i o n on keratinase (>150,000 MW) a l k a l i n e protease ( O ) , a keratinase ( © ) , and B keratinase ( A ) a c t i v i t y i n the absence (A) and presence (B) of added autoclaved feather solubles (AFS) m a t e r i a l . A c t i v i t y units = AOD 280 nm x 10 2/min/ml enzyme. 135 FIGURE 44. Comparison of the e f f e c t of d i l u t i o n of the >150,000 MW keratinase i n the presence of 5 mM (A) s u l f i t e , s o l i d l i n e ; b i s u l f i t e , dotted l i n e , (B) d i t h i o t h r e i t o l , and (C) cysteine on a l k a l i n e protease (0), a keratinase ( © ) , and 8 keratinase (A) a c t i v i t y . D i l u t i o n of the 150,000 MW keratinase i n the absence of a d d i t i o n a l ions (control) appears i n Figure 4 3A. A c t i v i t y units = AOD 280 nm 102/min/ml enzyme. 136 The e f f e c t of reducing agents on the molecular weight of the protease and  keratinase. To determine the e f f e c t of AFS and anions on the molecular weight of the keratinase, the enzyme was concentrated separately against PEG both i n the absence or presence of e i t h e r cysteine, DTT, or AFS, then chromatographed on Sephadex G100. As shown i n Figure 45, the e f f e c t of a l l the agents was to di s s o c i a t e the > 150,000 MW aggregate into 16,000 MW u n i t s . In the cases of cysteine and DTT, the a c t i v i t y of the a keratinase was maintained well i n the 16,000 MW peak. As previously mentioned, the phosphate-treated germination protease which eluted at 16,000 MW from G100 would not aggregate a f t e r d i a l y s i s against PEG or F i c o l l . However, when the phosphate-treated germination protease was reacted with 5 mM DTT, then dialysed against PEG i n the absence of DTT, and chromato-graphed on G100, the bulk of the enzyme protein eluted at > 150,000 MW (Figure 46A). Treatment of the phosphate-treated germination protease species with 5 mM DTT or 45 mM cysteine, then immediate G100 chromatography without concen-t r a t i o n against PEG, resu l t e d i n enzyme protein e l u t i o n at 16,000 MW with good a keratinase and c a s e i n o l y t i c a c t i v i t y (Figure 46B). K i n e t i c studies on the germination protease and keratinase. The k i n e t i c s of the germination protease and the phosphate-treated protease proved to be quite complex, as would be expected from the preceding r e s u l t s . K i n e t i c studies were conducted using denatured egg albumin and lysozyme as substrates. Keratin f r a c t i o n s were not used as substrates, because the 8 f r a c t i o n was insolu b l e i n the assay system and the a f r a c t i o n was i n s u f f i c i e n t l y characterized. Spore coat glycoproteins (Section III) also were not used as substrates because of low extr a c t i o n y i e l d s , i n s o l u b i l i t y of the Fraction B glycoprotein, and the previously noted low enzyme a c t i v i t y against these f r a c t i o n s . The following k i n e t i c data 137 M O L E C U L A R WE IGHT >150,000 49,000 16,000 i 1 1 4 6 8 10 12 14 16 18 20 22 24 F R A C T I O N N U M B E R FIGURE 45. E l u t i o n p r o f i l e s of the >150,000 MW keratinase a l k a l i n e protease (9) and a keratinase (O) a c t i v i t i e s a f t e r Sephadex G100 chromatography i n 0.05 M phosphate buffer pH 7.8. The samples were concentrated by d i a l y s i s against (A) PEG, (B) PEG + AFS, (C) PEG + 5 mM cysteine, and (D) PEG + 5 mM DTT before chromatography. The s o l i d l i n e represents protein e l u t i o n from the column. Enzyme a c t i v i t y = AOD 280 nm * 10 3/ min/ml f r a c t i o n s . M O L E C U L A R WEIGHT >150,000 67000 33,000 16,000 6 8 10 12 14 16 18 20 • F R A C T I O N N U M B E R FIGURE 46. Comparison of the e l u t i o n of the 16,000 MW phosphate-treated germination protease (from G100, phosphate buffer system) a f t e r (A) reduction with 5 mM DTT, then d i a l y s i s concentration against PEG and (B) reduction with 5 mM DTT, but without d i a l y s i s con-centration. A l k a l i n e protease ( © ) and a keratinase (O) a c t i v i t i e s were calculated as AOD 280 nm * 10 3/ min/ml f r a c t i o n . The s o l i d l i n e represents protein e l u t i o n from the column. 139 are presented as a preliminary report, describing a few of the very complex k i n e t i c plots obtained with t h i s enzyme. Heat or mercaptoethanol-denatured egg albumin served as an excellent sub-s t r a t e for protease a c t i v i t y . As shown i n Figure 47A, the p u r i f i e d germination protease (MW 33,000) displayed sigmoid k i n e t i c s with substrate i n h i b i t i o n on denatured albumin. Treatment of the protease with phosphate resulted i n an increased substrate threshold before a c t i v i t y was regained (Figure 47B). The i n h i b i t o r y e f f e c t of phosphate was removed at higher substrate concentrations (Figure 47A). A Lineweaver-Burk p l o t of the e f f e c t of phosphate on the germ-i n a t i o n protease gave a p l o t resembling competitive i n h i b i t i o n (dark l i n e s , Figure 48). At lower substrate concentrations, however, the r e s u l t s could be p l o t t e d to give more than one point of i n t e r s e c t i o n ( l i g h t l i n e s , Figure 48), and hence more than one K i per i n h i b i t o r concentration. This r e s u l t was not e n t i r e l y unexpected, considering the sigmoid k i n e t i c s noted previously. A re p l o t of the data from Figure 48, gave a p l o t e x h i b i t i n g hyperbolic competitive i n h i b i t i o n (Figure 49). These r e s u l t s suggested that phosphate acted on the enzyme at a s i t e d i f f e r e n t from the substrate-binding s i t e , and that phosphate affe c t e d the enzyme's a f f i n i t y f o r the substrate, but not the rate of product formation. Lysozyme also proved to be a useful substrate for k i n e t i c studies. The germination protease (33,000 MW) exhibited marked substrate i n h i b i t i o n on this substrate (Figure 50). Phosphate treatment of the enzyme resulted i n perfect sigmoid k i n e t i c s at I O - 2 M and 10" 3 M phosphate concentrations. As the e f f e c t of phosphate treatment was to increase the substrate threshold i n denatured albumin substrate, i t was assumed that the control enzyme on lysozyme also d i s -played sigmoid k i n e t i c s , but the substrate threshold was attained at a much lower concentration than could be measured using t h i s assay system. The k i n e t i c & • 1 1 I I 10~ 6 5X10" 6 10~ 5 5x10~ 5 10 " 4 (M) SUBSTRATE CONCENTRATION FIGURE 47. Sigmoid k i n e t i c s of the 3 hr germination protease on denatured albumin substrate (0.05 M Veronal buffer, pH 9.0) i n the absence ( © ) and presence ( O ) of 1 0 - 3 M inorganic phosphate (A). B shows an i n -crease i n the 3 hr germination protease ( © ) substrate binding threshold i n the presence of 10" 2 M ( O ) , IO" 3 M ( H ) , and 10-1* (•) inorganic phosphate. V e l o c i t y was c a l c u l -ated as AOD 280 nm/min/mg enzyme prot e i n . 141 /S (M) FIGURE 48. A double r e c i p r o c a l plot of the rate of product formation by 3 hr germination protease i n the absence ( © ) and presence o f I O - 2 M ( O ) , 10* 3 M ( • ) , and 10 - 1 + M ( A ) inorganic phosphate on denatured albumin substrate (0.05 M Veronal b u f f e r , pH 9.0). V e l o c i t y was c a l c u l a t e d as AOD 280 nm/min/mg enzyme protein. 142 FIGURE 49. Plot of 1/v against inhibitor concentration showing hyperbolic competitive inhibition of the 3 hr germination protease by inorganic phosphate in 2.5 x IO-1* M (®), 5 x 10~5 M (O) , 5 x IO - 6 M (©) and 2.5 x 10"6 M ( Q ) albumin substrate concentrations. (0.05 M Veronal buffer, pH 9.0). The value for 1/v was calculated from the double reciprocal plot in Figure 48. The arrow indicates decreasing substrate concentration. Velocity was calcul-ated as AOD 280 nm/min/mg enzyme protein. 143 ) T — l 1 1 : r e 1 1 1 i i i _ _ 5x1CT 6 1CT5 5x10" 5 10 " 4 ( M ) SUBSTRATE CONCENTRAT ION FIGURE 50. Rate p l o t of the 3 hr germination protease on lysozyme substrate ( © ) and the e f f e c t of 1 0 - 2 M ( O ) , 1 0 - 3 M ( • ) , and 10~ u M ( ® ) inorganic phosphate on the co o p e r a t i v i t y of substrate binding (0.05 M Veronal buffer pH 9.0). V e l o c i t y was c a l c u l a t e d as AOD 280 nm/min/mg enzyme prot e i n . 144 curve i n the presence of 10 M phosphate, however, was l i n e a r i n d i c a t i n g that at an optimal phosphate concentration the negative cooperativity of substrate-binding was abolished and that phosphate acted as an a c t i v a t o r as well as an i n h i b i t o r . This r e s u l t was i n agreement with the data i n Figures 35, 36, and 37, where inorganic phosphate also appeared as an a c t i v a t o r . Comparison of the k i n e t i c curves f o r the 33,000 MW germination protease treated with phosphate (IO - 1* M) and the keratinase showed that both enzymes displayed s i m i l a r l i n e a r k i n e t i c s with some substrate i n h i b i t i o n (Figure 51). Although a d e t a i l e d k i n e t i c analysis of the e f f e c t of reducing agents on the keratinase or the phosphate-treated protease was not attempted, some inform-at i o n was obtained by comparing the urea-denaturation curves f o r the germination protease and the keratinase (16,000 MW). As shown i n Figure 52, the keratinase was much more s e n s i t i v e to urea than the germination protease. Treatment of the protease with phosphate gave a denaturation curve s i m i l a r to that of the protease alone, but with lower a c t i v i t y , as would be expected. Treatment of the germination protease with phosphate,then with reducing agents ( t h i o s u l f a t e , s u l f i t e , b i s u l f i t e ) gave a denaturation curve i d e n t i c a l to that of the keratinase. The e f f e c t of metal ions on germination protease a c t i v i t y . In a survey of the e f f e c t s of cations on the germination protease, i t was found that 10" 3 M ethylene diamine t e t r a a c e t i c a c i d (EDTA) added to germinating spores (10 5/ml) i n h i b i t e d germination by 50%. As shown i n Figure 53A, manganese and magnesium ions stimulated germination protease a c t i v i t y . These ions also stimulated spore germination. Copper, n i c k e l and s i l v e r ions were i n h i b i t o r y to both enzyme a c t i v i t y and spore germination. Zinc and ir o n ions i n h i b i t e d germination, but stimulated protease a c t i v i t y . D i l u t i o n of the germination protease resulted i n decreased enzyme a c t i v i t y (Figure 53B). Both magnesium and manganese prevented a c t i v i t y loss due to d i l u t i o n , and manganese proved to be quite stimulatory to 5x10"6 IO"5 5x10"5 10" 4 5x10"4 10"3 (M) SUBSTRATE CONC. FIGURE 51. Comparison of the rate of lysozyme substrate u t i l i z a t i o n by the 16,000 MW keratinase ( © ) and the 3 hr germination protease + 10-t* M inorganic phosphate ( O ) . The 3 hr germination protease without a d d i t i o n a l phosphate was used as control ( • ) . (0.05 M Veronal buffer, pH 9.0). The rate was ca l c u l a t e d as AOD 280 nm/min/mg enzyme pro t e i n . 146 T 1 1 1 1 1 r © MOLAR UREA FIGURE 52. Comparison of the s e n s i t i v i t y of the germination protease ( © ) and the keratinase ( O ) to urea i n a c t i v a t i o n . Treatment of the germination protease with I O - 5 M inorganic phosphate (•) or I O - 3 M t h i o s u l f a t e ( A ) decreased the enzyme's a c t i v i t y i n the presence of urea. (0.05 M Veronal bu f f e r pH 9.0). A c t i v i t y was calculated as AOD 280 nm/min/mg enzyme pr o t e i n . Relative a c t i v i t y was cal c u l a t e d as a c t i v i t y i n the presence of urea * a c t i v i t y without urea. 147 0 10"2 10"3 10" 4 10" 5 10- 6 10- 7 10" 8 O 10 20 30 40 (M) ION CONCENTRAT ION DILUTION FACTOR FIGURE 53. A. Comparison of the e f f e c t of metal ions on the germination protease a c t i v i t y . The e f f e c t of copper ions ( A ) , calcium ions ( A ) , manganese ions ( • ) , and magnesium ions ( O ) are shown r e l a t i v e to the con t r o l without a d d i t i o n a l ions (•) i n 1% casein substrate i n 0.05 M Veronal b u f f e r pH 9.0. B. Prevention of loss of germination protease enzyme a c t i v i t y with d i l u t i o n (•) by I O - 5 M manganese ions ( © ) and I O - 5 M magnesium ions ( O ) . A c t i v i t y was ca l c u l a t e d as AOD 280/min/mg enzyme protein. 148 lower enzyme concentrations. Reactivation of the phosphate-treated germination protease with magnesium or manganese, however, was unsuccessful. As shown i n Table 25, the 16,000 MW keratinase's a keratinase a c t i v i t y was act i v a t e d by magnesium, manganese, cobalt, and zinc cations. Generally, greater a c t i v a t i o n of the a l k a l i n e protease a c t i v i t y of this keratinase was obtained using the same ions (except magnesium). TABLE 25. Comparison of the e f f e c t of cations on the a keratinase and a l k a l i n e protease a c t i v i t y of the 16,000 MW keratinase C a t i o n 3 Related S p e c i f i c A c t i v i t y b a k e r a t i n a s e 0 a l k a l i n e protease^ 10" 3 M 10" 5 M IO" 3 M 10~ 5 M ions ions ions ions control 1.0 1.0 1.0 1.0 Mg 2.48 1.92 1.0 1.0 Mn 2.0 3.4 5.0 4.0 C o " 1.08 1.6 6.0 6.67 Ag + 0.68 1.0 0 0 Zn 2.8 1.6 6.0 6.67 C a ^ 0 1.08 2.33 1.67 C u ^ 1.2 1.0 1.73 1.0 N i + 0.08 0.08 1.2 0.53 3 a l l cations are chloride s a l t s , made up i n the appropriate assay b u f f e r b S p e c i f i c a c t i v i t y = AOD 280 * 103/min/mg enzyme protein c a k e r a t i n substrate i n 0.05 M Veronal buffer pH 7.0 ^ casein substrate i n 0.05 M Veronal buffer pH 9.0 150 Discussion: In order for s i g n i f i c a n t hydrolysis of native k e r a t i n to occur, the outer b a r r i e r of the k e r a t i n i z e d c e l l membranes must be penetrated. These membranes are composed of 8 k e r a t i n which r e s i s t s extreme conditions (exposure to 5N NaOH, 8 M urea, 8 M urea + t h i o g l y c o l l i c a c i d at pH 10, or 10% sodium s u l f i t e ) but are susceptible to p r o t e o l y t i c enzymes l i k e t r y p s i n (Mercer, 1961). The inner com-ponent of these c e l l s , the a k e r a t i n f i b r i l s , r e s i s t t r y p s i n action, but are s e n s i t i v e to the other treatments, g i v i n g k e r a t i n a "complementary" structure. The other b a r r i e r to complete h a i r digestion i s the high concentration of d i -s u l f i d e bonds which c r o s s - l i n k the a k e r a t i n f i b r i l s and the 8 k e r a t i n membranes. Digestion of k e r a t i n by dermatophytes i s accompanied by the elaboration of complex "eroding mycelia", which promote penetration and erosion of the h a i r structure (English, 1962). These structures have been shown by Kunert (1972) to produce s u l f i t e which reduces the k e r a t i n d i s u l f i d e s by " s u l f i t o l y s i s " according to the r e a c t i o n : R-S-S-R + HS0| R-SH + RS-SOf and t h i s reduction, i n turn, exposes the k e r a t i n proteins to enzymatic attack. A s i m i l a r reduction of d i s u l f i d e s p r i o r to k e r a t i n o l y t i c attack has been ob-served i n the digestion of wool by the clothes moth (Tineola b i s s e l l i e l l a ) l a r v a (Powning, 1956; Powning & Irzykiewicz, 1960). It appears reasonable that the germination protease would possess 8 kera-t i n o l y t i c a c t i v i t y , as germinating spores on k e r a t i n substrates would encounter the 3 k e r a t i n f i r s t . The chymotrypsin-like preference of the germination pro-tease f o r tyrosine and aromatic amino acids has been reported previously (Leighton & Stock, 1970a; 1970b). The resistance of the 8 k e r a t i n i s thought to be due to c r o s s - l i n k i n g between tyrosine residues, so treatments which are active against tyrosine would s o l u b i l i z e the 8 k e r a t i n f r a c t i o n (Alexander & Hudson, 1954). 151 Limited 8 k e r a t i n digestion may expose d i s u l f i d e linkages which then induce s u l f i t e formation and production of the "eroding mycelium" complex. Released s u l f u r compounds may play an a d d i t i o n a l r o l e i n stimulating the polymorphic changes necessary for eroding mycelium formation, as i t has been shown that s u l f u r amino acids (methionine) stimulate the growth and dimorphism of the i n -vasive form of Candida albicans (Mardon, 1969; 1971). Mechanical penetration of the outer b a r r i e r of 8 k e r a t i n membranes would then expose the a k e r a t i n f i b r i l s to further k e r a t i n o l y t i c action. This would require the keratinase to be tolerant of a d i s u l f i d e - r e d u c i n g environment and to be active against a k e r a t i n . This keratinase would be derived most economically from the germin-ati o n protease, which i s the only protease released on spore germination and outgrowth (Leighton & Stock, 1970a). The r e s u l t s suggested that the germination protease underwent a t r a n s i t i o n i n a c t i v i t y f o r substrate a f t e r germination and phosphate " i n a c t i v a t i o n " . Where the phosphate acted as an i n h i b i t o r of c a s e i n o l y t i c , spore coat p r o t e o l y s i s , and BTEE esterase a c t i v i t y , i t also acted as an a c t i v a t o r of a k e r a t i n o l y t i c a c t i v i t y . Other e f f e c t s of phosphate on the germination protease were the change i n pH optimum from pH 9 to pH 7, and the reduction i n molecular weight from 33,000 to 16,000. A s i m i l a r phosphate-induced d i s s o c i a t i o n of chymotrypsin has been re-ported previously (Tinoco, 1957). The lower molecular weight and pH optimum corresponded to those of the keratinase formed when M. gypseum was grown on k e r a t i n as sole carbon and nitrogen source. The pH optimum was also i n agree-ment with that found f o r the keratinase of Trichophyton mentagrophytes (Yu, Harmon & Blank, 1969). The r e s u l t s suggested, however, that the 16,000 MW unit formed a f t e r phos-phate treatment of the germination protease did not have the same molecular properties as the keratinase, as the former would not aggregate when concentrated 152 by d i a l y s i s . A f t e r d i s u l f i d e reduction, however, the 16,000 MW unit did form aggregates and had enhanced a k e r a t i n o l y t i c a c t i v i t y . This observation was consistent with the preceding model, which suggested that the a keratinase must t o l e r a t e a di s u l f i d e - r e d u c i n g environment, and thus would e x i s t most l i k e l y i n a sulfhydryl-reduced s t a t e . A summary of these protease-keratinase molecular interconversions i s pre-sented i n Figure 54. The i n t r a c e l l u l a r events were derived from Table 21. The i n t r a c e l l u l a r 16,000 MW protease which appeared a f t e r 2 days sporulation was probably the same as the a keratinase which was released e x t r a c e l l u l a r l y . This p o s s i b i l i t y was supported by the fac t that both of these enzymes w i l l aggregate to form large complexes, and that a keratinase was detected i n mycelial extracts immediately a f t e r protease induction ( F i g . 40B) and p r i o r to germination protease formation (Fig. 40A). Recently, i t was shown that a mutant s t r a i n (B6) of M. gypseum which was defective i n spore formation beyond the 2 day stage, possessed active a keratinase, but neither a l k a l i n e protease nor 8 keratinase. (N. Wong, unpublished data). The aggregates formed by the keratinase possibly a r i s e through non-specific a s s o c i a t i o n , but the fac t that these complexes were active suggested that they were assembled i n a s p e c i f i c manner. When the molecular weights of the commonly occurring complexes were examined, i t was apparent that a l l the complexes were composed of even multiples of 8,000 MW and sequential multiples of 16,000 Mw (Table 26). I f the re s u l t s reported i n Table 22 did represent a sequential synthesis and polymerization of the germination protease during sporulation, then the 8,000 MW unit may be assumed to be the smallest protomer of the 33,000 MW germination protease and of the 16,000 MW keratinase. The bonds holding the 8,000 MW protomers together i n the 16,000 MW unit were probably d i f f e r e n t from the bonds un i t i n g the two 16,000 MW uni t s , as 153 SPORE GERMINATION INTRACELLULAR U 8,000 MW >150,000MW i16,000MW i 33,000MWOO 16,000 MW 0 4 keratinase activity VEGETATIVE GROWTH 00 pkeratinase activity 33,000 MW P0 4=(a)-J SOo= 16,000MW •<keratinase activity - i dialysis OO 33.OO0MW p keratinase activity 16.000MW enhanced « keratinase activity > sssmu >150,000MW FIGURE 54. Summary of the proposed M. gypseum protease-keratinase molecular interconversions. Phosphate may be (a) re-leased by germinating spores or (b) released by the growing hyphae (M. Kavanaugh, unpublished data). Further d e t a i l s appear i n the discussion. 154 conditions which caused d i s s o c i a t i o n of the 33,000 MW germination protease did not y i e l d 8,000 MW protomers. Furthermore, the 8,000 MW protomer was observed only i n early (2 day) sporulating mycelial extracts, and i t was not possible to d i s s o c i a t e active 8,000 MW protomers from the 16,000 MW units. The pleomorphic s t r a i n , a f t e r 3 days aeration, however, may prove to be a good source of the 8,000 MW unit for further studies. Using the terminology of Monod (1965), the 16,000 MW unit was probably composed of two 8,000 MW protomers bonded by isologous associations (where the domain of bonding involved two i d e n t i c a l binding s e t s ) . An isologous dimer can be further polymerized to give r i s e to even numbered oligomers. The 16,000 MW u n i t s , however, may have aggregated by heterologous a s s o c i a t i o n , where the domain of bonding had no element of symmetry, and large polydisperse polymers arose. The r e s u l t s suggested that d i s u l f i d e bonds (possibly random) were i n -volved i n keratinase aggregate formation, as d i s u l f i d e reducing agents d i s s o c i -ated these aggregates into 16,000 MW units (Figure 45). This model, therefore, would allow aggregates composed of even multiples of 8,000 MW and sequential multiples of 16,000 MW to a r i s e (Table 26). The r e s u l t s reported here are l i m i t e d by the nature of the protease assay employed. Although the assay supernatant f l u i d , a f t e r PCA p r e c i p i t a t i o n con-tained a l l the amino acids and short peptides released by the protease or keratinase, e s s e n t i a l l y only the aromatic amino acids released by p r o t e o l y s i s were measured by estimating the AOD 280 nm. This condition implies two things about the data: (a) the substrates were hydrolysed best when aromatic amino acid linkages were r e a d i l y a v a i l a b l e to p r o t e o l y t i c attack, and (b) low substrate u t i l i z a t i o n probably r e f l e c t e d low aromatic amino acid release. In the l a t t e r case, there may have been considerable amino acid or peptide release, but these products possibly were not detected by measuring AOD 280 nm. The protease assay TABLE 26. Evidence for isologous and heterologous a s s o c i a t i o n of the keratinase subunits Possible T h e o r e t i c a l Molecular Weight Experimental Molecular Weight 3 Number of 8,000 MW Protomers Number of 16,000 MW subunits 8,000 16,000 24,000 32,000 40,000 48,000 52,000 64,000 124,000 8,000b 16,000 33,000 49,000 66,000 120,000 8 16 4 8 3 by Sephadex G100 and G200 column chromatography b found i n 1 day o l d s t r a i n R87 hyphae only 156 could be made more s e n s i t i v e to general amino a c i d release by performing an amino nitrogen (ninhydrin) t e s t on the assay supernatant f l u i d (Matheson & T a t t r i e , 1964). However, when Yu, Harmon, and Blank (1969) compared keratino-l y t i c a c t i v i t y by amino nitrogen release and by AOD 280 nm, very l i t t l e d i f f e r -ence i n e i t h e r the rate of substrate hydrolysis or the pattern of hydrolysis was obtained. Measurement of protein and peptides released by p r o t e o l y s i s also has been employed by McDonald and Chen (1965). However, the F o l i n reagent gave the greatest amount of colour development with aromatic amino acids (Lowry et a l , 1951; Chou & Goldstein, 1960), so t h i s modification tended to increase the number of assay tubes and the experimental error. Another great drawback to t h i s method was that accidental contamination of the protease assay super-natant f l u i d with PCA p r e c i p i t a t e material gave erroneously high protein values. Measurement of enzyme a c t i v i t y by the AOD 280 nm method was deemed correct, however, as previous authors had used this method with success (Leighton & Stock, 1970a; 1970b; Yu, Harmon & Blank, 1969) and the a l k a l i n e protease of M» gypseum was determined to possess s p e c i f i c i t y f o r aromatic linkages (Leighton & Stock, 1970b). Also, as both the a l k a l i n e protease and the keratinase were affec t e d by f reeze—thawing (Figure 42 & Table 23) and d i l u t i o n (Figure 43 & Figure 53), r e p r o d u c i b i l i t y of enzyme a c t i v i t y values from day to day was d i f -f i c u l t to obtain. To counteract these d i f f i c u l t i e s , a l l the assays where comparisons were drawn, were conducted on the same day. As t h i s often re-quired 200 or more protease assays to be executed i n one day, the measurement of protease a c t i v i t y by AOD 280 nm was decided to be the fas t e s t and most s e n s i t i v e method a v a i l a b l e . One also must viexj the k i n e t i c s ' r e s u l t s with some caution. As the sub-strates of the germination protease and keratinase were polymeric proteins i n vi v o , complex protein substrates were used to attempt to determine how these 157 enzymes would react i n a natural s i t u a t i o n . A f t e r cleavage of one or more peptide bonds, however, the substrate was no longer a s i n g l e p r o t e i n , and multiple substrates were generated. This problem would be eliminated i f synthetic mono-peptides were used, but then, very l i t t l e would be learned about the enzyme's a b i l i t y to hydrolyse complex proteins. The discussion of the k i n e t i c data, therefore, i s l i m i t e d to studies using complex natural protein substrates, i n an e f f o r t to elucidate the mechanisms of hydrolysis of complex proteins by the M. gypseum germination protease and keratinase. Phosphate acted as e i t h e r an a c t i v a t o r or i n h i b i t o r of protease a c t i v i t y , conforming with Monod's model f o r a l l o s t e r i c enzyme systems (Monod et a l , 1965). As shown i n Figure 50, I O - 2 M and 1 0 - 3 M phosphate concentrations acted as i n -h i b i t o r s , increasing the threshold of the substrate saturation curve and d i s -placing the Michaelis substrate constant to the r i g h t , while the a c t i v a t o r (10 - 1* M phosphate) abolished the threshold of substrate-binding and displaced the Michaelis constant to the l e f t . These types of curves were c h a r a c t e r i s t i c of homotropic cooperative e f f e c t s , where the substrate also acted as an a l l o -s t e r i c ligand (Monod. et a l , 1965). Another a l l o s t e r i c e f f e c t of substrate concentration, observed i n a l l the substrates used, was substrate i n h i b i t i o n . This type of i n h i b i t i o n was obtained when ES = EI and SES = IES, or the binding of substrate molecules to a l l o s t e r i c s i t e s reduced the rate of product formation (Mahler et a l , 1966). As shown i n Figure 49, phosphate was a hyperbolic competitive i n h i b i t o r of germination protease a c t i v i t y . This type of i n h i b i t i o n caused an increase i n the Michaelis constant of the substrate. An EI complex did not form and I did not compete with S for enzyme-binding, as i n competitive i n h i b i t i o n , but an IES complex was formed and was converted to products at the same rate as the ES complex. The e f f e c t of i n h i b i t o r was, therefore, only on the a f f i n i t y of the 158 enzyme f o r substrate-binding (Mahler et a l . 1966). This type of i n h i b i t i o n has been shown previously to occur i n NAD-inhibited NADH oxidase (Worcel et a l . 1965). These r e s u l t s also indicated that phosphate may bind at an a l l o s t e r i c s i t e d i f f e r e n t from the substrate-binding s i t e . One could imagine that the protease s p e c i f i c substrate i n the spore coat possessed very s p e c i f i c linkages, hydrolysis of which yielded maximum c e l l w all p l a s t i c i t y with minimal w a l l polymer destruction. The subunit construction and interprotomer bonding found i n the germination protease could ensure that the substrate-binding s i t e was constrained into a very precise conformation. This complex molecular architecture would also e f f e c t i v e l y prevent indiscriminate ass o c i a t i o n of the germination protease subunits or protomers with other c e l l proteins to give an act i v e molecule (Monod et a l , 1966). I t i s not unreasonable to expect that the enzyme c o n t r o l l i n g M. gypseum spore germination should be under these s t r i c t controls, as accidental germination, cleavage of the wrong substrate and over-hydrolysis of the spore coat could be disastrous to the spore's s u r v i v a l . The r a t e - l i m i t i n g step which controls B a c i l l u s cereus endospore germination (Halvorson et a l , 1966; Wolgamott & Durham, 1971) and the cont r o l of yeast bud formation ( K e l l e r & Cabib, 1971) s i m i l a r l y have been shown to be under the control of a l l o s t e r i c enzymes. The keratinase, on the other hand, should possess general p r o t e o l y t i c a c t i v i t y and should cause maximum protein destruction i f i t i s to function as an e f f i c i e n t e x t r a c e l l u l a r ( n u t r i t i o n a l ) enzyme. The substrate s p e c i f i c i t y of the M. gypseum keratinase has not been studied using synthetic peptides, but the keratinase of T. mentogrophytes has been shown to function as an endopeptidase and an exopeptidase, cleaving 25 amides, 21 peptides and 9 proteins (Yu et a l , 1969). The keratinase of Streptomyces fradiae s i m i l a r l y was shown to hydrolyse a wide v a r i e t y of peptides and proteins (Nickerson & Durand, 1963). 159 The keratinase of M. gypseum exhibited l i n e a r k i n e t i c s on lysozyme sub-s t r a t e . The phosphate-binding s i t e s of the germination protease may be associated with the esterase a c t i v e s i t e , which was shown to be s e n s i t i v e to phosphate and PMSF i n h i b i t i o n . In this case, the phosphate possibly was bound through serine residues (Oosterbaan & Cohen, 1964). At higher phosphate con-centrations, the phosphate possibly d i s s o c i a t e d the 33,000 MW oligomer by p r e c i p i t a t i o n of the metal ions holding the 16,000 MW subunits together. (Alder et a l , 1939; Lotspeich & Peters, 1951). The resultant 16,000 MW units then were rendered more active against keratins by d i s u l f i d e reduction and, presumably, a c o n f i g u r a t i o n a l change ensued a f t e r release of the constraints imposed by the d i s u l f i d e bridges (Boyer, 1959). Hydrogen bonding or hydro-phobic bonding may be involved i n the enzyme's new configuration, as evidenced by the denaturant e f f e c t s of urea i n Figure 52 (Bruning & Holtzer, 1961; Kauzmann, 1961; Linderstr^m-Lang et a l , 1959). The r e s u l t s also showed that t h i o s u l f a t e , the product of s u l f i t o l y s i s , stimulated keratinase a c t i v i t y , as did cysteine, a possible k e r a t i n hydrolysis product. 160 General Discussion: The r e s u l t s of t h i s i n v e s t i g a t i o n have shown that the l y t i c events i n Microsporum gypseum macroconidium germination were i n i t i a t e d by the action of an a l k a l i n e protease. The a c t i v i t y of t h i s protease was regulated by i n -organic phosphate, concomitantly released from germinating spores, such that the number of spores germinating within a given population or given volume of germination medium was r e s t r i c t e d . For t h i s reason the amount of a c t i v e enzyme which could be c o l l e c t e d from germinating spores was l i m i t e d , as i n -creased spore densities i n the germination medium decreased the number of spores germinating and also decreased the expected y i e l d of active germination protease. The mechanism of a l k a l i n e protease release during spore germination was found to be v i a lysosomal v e s i c l e s , formed i n t r a c e l l u l a r l y during sporulation and in s e r t e d i n t o the spore coat during spore maturation. Vesicles of two types were found i n the spore coats: (a) those present i n ungerminated spores (electron-transparent) and (b) those found i n germination-initiated spores (electron-dense). The v e s i c l e s of type (a) were found to contain large amounts of a l k a l i n e protease a c t i v i t y , and the v e s i c l e s of type (b) contained large amounts of a l k a l i n e phosphodiesterase a c t i v i t y . The r e s u l t s suggested that these v e s i c l e s o r i g i n a t e d from the endoplasmic reticulum. The v e s i c l e s pos-s i b l y were inse r t e d into the spore coat by reverse pinocytosis (Gibson & Peberdy, 1972) or more l i k e l y by fusion of m u l t i v e s i c u l a r v e s i c l e s with the plasmalemma (Marchant & Robards, 1968; Gu l l & T r i n c i , 1971; Takeo et a l , 1973). The r e s u l t s suggested that the macroconidia of M. gypseum were capable of germination as soon as they were introduced into a s u i t a b l e medium. Spore coat hydrolases probably were released from lysosomes a f t e r water imbibition and sub-sequent v e s i c l e rupture. The compartmentalization of germination hydrolases i n 161 v e s i c l e s made pos s i b l e d i s c r e t e l o c a l i z a t i o n of these enzymes at one or more places within the spore coat. The hydrolysis of the spore coat upon germin-ation could, therefore, be l o c a l i z e d rather than general, weakening a small portion of the spore coat rather than the e n t i r e s t r u c t u r e , and possibly deter-mining the shape of the germ tube. Such a mechanism would eliminate the need for a s p e c i f i c "plug" i n the spore coat, hydrolysis of which would provide a passage for the germ tube (Skucas, 1966). After germ tube i n i t i a t i o n , however, the germination protease was released i n t o the germination medium. The prote-ase, at t h i s time, could attack the e n t i r e spore coat i n a general, nonspecific manner, thus n e c e s s i t a t i n g protease i n a c t i v a t i o n . Although the a l k a l i n e protease appears to be the hydrolase which i n i t i a t e d M. gypseum macroconidium germination, the e n t i r e process of spore coat weakening to allow germ tube emergence was seen to be the r e s u l t of the sequential action of several hydrolases. The f i r s t enzymes a c t i v e i n the spore coat were a l k a l i n e protease and 61,3 glucanase. These enzymes were followed by a l k a l i n e phospho-diesterase, BTEE esterase, and N-acetyl glucosaminidase a c t i v i t i e s . The timing of a l k a l i n e protease a c t i v i t y preceding a l k a l i n e phosphodiesterase a c t i v i t y was important, as t h i s sequence of a c t i v i t y allowed germination i n i t i a t i o n to pre-cede inorganic phosphate release and concomitant a l k a l i n e protease i n a c t i v a t i o n . The r e s u l t s of t h i s i n v e s t i g a t i o n suggested that the macroconidia of M. gypseum were formed as a r e s u l t of a l t e r e d hyphal t i p elongation, which i n -volved considerable a l t e r a t i o n s of the plasmalemma properties and c e l l w all composition and st r u c t u r e . Macroscopically, sporulation was indicated by cessation of a p i c a l t i p elongation, with continued septation and increased c e l l w all thickening. Chemically, M. gypseum macroconidia coats contained greater N-acetyl glucosamine and p r o t e i n than hyphal walls (Leighton & Stock, 1970a). Observations obtained during work i n t h i s thesis have shown that the 162 macroconidial coats contained greater carbohydrate-protein ester, d i s u l f i d e bridge, acid-extractable phosphate, and acid-nonextractable phosphate than mycelial walls. The protein of the spore coats was accounted for completely as the sum of extractable glycoprotein-protein and nonextractable r e s i d u a l p r o t e i n . The germination protease did not display a c t i v i t y against the spore coat r e s i d u a l protein. The germination protease s p e c i f i c substrate within the spore coat was found to be the water-insoluble glycoprotein, which was released by ethylene diamine extraction (Korne & Northcote, 1960). This spore coat component was found to be a major constituent of the spore coats, and underwent s p e c i f i c synthesis at the time of sporulation. I f the water-insoluble glycoprotein was derived from i t s hyphal counterpart, considerable a l t e r a t i o n of t h i s f r a c t i o n ' s protein and amino acid content had occurred during sporulation. The high content of c h i t i n and glucan " c r o s s - l i n k i n g " amino acids: a s p a r t i c a c i d , glutamic acid, serine, and threonine (Sentrandreu & Northcote, 1968) indi c a t e d that t h i s glycoprotein f r a c t i o n may have contributed to the s t r u c t u r a l s t a b i l i t y of the spore coat. The water-insoluble glycoprotein also contained considerable quantities of aromatic amino acids, which would make t h i s f r a c t i o n susceptible to a l k a l i n e germination protease attack (Leighton & Stock, 1970b). The other glycoprotein constituent of the spore coat, extractable with ethylene diamine, was considered not to be involved d i r e c t l y with sporulation, as i t was found to be present i n equal quantities and was of s i m i l a r composition i n both the mycelial, walls and the spore coats. This water-soluble glycoprotein, however, was found to be susceptible to a l k a l i n e protease h y d r o l y s i s , and was found to be turned-over as a carbohydrate and nitrogen reserve material, i n stages of advanced st a r v a t i o n . I n h i b i t o r s of macromolecular synthesis, commonly used to study the 163 p h y s i o l o g i c a l changes associated with germination, have been divided i n t o two cla s s e s : (a) those e f f e c t i v e p r i o r to germination i n i t i a t i o n and (b) those e f f e c t i v e a f t e r germ tube emergence. The time when i n h i b i t o r s are e f f e c t i v e may r e f l e c t differences i n i n t r a c e l l u l a r synthetic capacities (Van Etten, 1969) or may r e f l e c t differences i n the penetration or exclusion of the i n h i b i t o r , as a r e s u l t of a l t e r a t i o n s i n c e l l surface properties and permeabilities. ( N i s h i , 1961; Sussman & Halvorson, 1966). The r e s u l t s of t h i s i n v e s t i g a t i o n suggest that the c e l l surface as well as the spore coat i n t e r n a l structure were alt e r e d considerably during M. gypseum macroconidium germination and outgrowth. These observations demonstrate the absolute requirement for proof of i n h i b i t o r uptake in t o the c e l l before conclusions on the synthetic po t e n t i a l of ungerminated spores can be made with c e r t a i n t y . For example, several authors have concluded that the ungerminated spores of B l a s t o c l a d i e l l a emersonii, Botrydiplodia  theobromae, Peronospora tabacina, and Uromyces phaseoli contain preformed and conserved messenger RNA, which i s necessary f o r early protein synthesis during germination. This conclusion i s based on the i n a b i l i t y of Actinomycin D to i n h i b i t i n i t i a l germination, although i t has not been shown that t h i s a n t i b i o t i c a c t u a l l y penetrates the ungerminated spore (Lovett, 1968; Ramakrishnan & Staples, 1968; Brambl & Van Etten, 1970; Holloman, 1971). The i n h i b i t o r y action of inorganic phosphate on the a c t i v i t y of the germ-i n a t i o n protease was found to be very complex. The immediate e f f e c t of i n -organic phosphate was to i n h i b i t the esterase and c a s e i n o l y t i c a c t i v i t y of the germination protease. This acti o n , presumably was responsible for c o n t r o l l i n g the degree of spore coat hydrolysis during germination, preventing over-hydrolysis of the spore coat and l y s i s of the germling. The action of the inorganic phos-phate was not e n t i r e l y i n h i b i t o r y , however, as the germination protease was re-tained as an a c t i v e e x t r a c e l l u l a r enzyme, but had a l t e r e d s p e c i f i c i t y f o r 164 substrate, a lowered pH optimum, and a decreased molecular weight. The sub-s t r a t e favoured a f t e r inorganic phosphate reaction with the germination pro-tease was k e r a t i n . The probable homology between the e x t r a c e l l u l a r k e r a t i n -ase of M. gypseum and the inorganic phosphate-reacted germination protease was demonstrated by i n v i t r o conversion of the germination protease i n t o a keratinase-possessing s i m i l a r properties to those of the e x t r a c e l l u l a r k e r a t i n -ase induced i n the presence of exogenous ke r a t i n substrates. Further proof of the o r i g i n of the keratinase from the a l k a l i n e protease may be obtained with a comparison of t o t a l amino acid composition data, N-terminal and C-terminal amino acids, and t r y p t i c peptide " f i n g e r - p r i n t s " (Shoer & Rappaport, 1972). The keratinase generated by inorganic phosphate reaction with the germin-ation protease appears to be a subunit (16,000 MW) of the l a t t e r enzyme (33,000 MW). Subunit construction of the germination protease also was suggested by an apparent sequential aggregation of increasingly large (8,000-33,000 MW) a l k a l i n e protease complexes during sporulation. The germination protease was found to possess a c t i v i t y against the 8 - f r a c t i o n of h a i r k e r a t i n , or the h a i r surface pro t e i n . The 16,000 MW keratinase was found to be most ac t i v e against the i n t e r n a l o t-keratin f i b r i l s , which comprise the i n t e r i o r of the h a i r structure. Other authors have reported that a number of enzymes are involved i n k e r a t i n digestion (Yu, Harmon & Blank, 1968; 0'Sullivan & Mathison, 1971) and Yu et a l (1971) have suggested that the i n t r a c e l l u l a r and e x t r a c e l l u l a r keratinases of Trichophyton mentagrophytes were r e l a t e d , as they cross-reacted immunologically. Derivation of the keratinase enzyme from the spore germin-ation or vegetative a l k a l i n e protease, however, has not been proposed previously. Keratinase a c t i v i t y was found to be enhanced by d i s u l f i d e reducing agents, by cysteine, and by s u l f u r oxidation products ( t h i o s u l f a t e ) . This f i n d i n g sup-ported the observation that s u l f i t e reduction of keratin d i s u l f i d e s ( " s u l f i t o l y s i s " ) 165 may precede p r o t e o l y t i c hydrolysis of keratins (Kunert, 1972). The release of s u l f i t e probably was r e s t r i c t e d to the area immediately surrounding the hyphal "penetration complex", which may explain why Chesters and Mathison (1963) were unable to detect s u l f u r reduction products during Keratinomyces a j e l l o i k e r a t i n o l y s i s . The dry surface environment which a dermatophyte would encounter i n a s u p e r f i c i a l dermatomycosis s i t u a t i o n , would necessitate the s p e c i f i c , l o c a l i z e d elaboration and release of d i s u l f i d e reducing agents and keratinases. This inhospitable environment also would necessitate the l e a s t expensive and most rapid switch from a spore germling outgrowth mode of a c t i v i t y to an invasion and exogenous substrate u t i l i z a t i o n mode of a c t i v i t y . The derivation of the keratinase from the germination protease, mediated by factors which control spore germination (inorganic phosphate) and by factors which precede k e r a t i n -o l y s i s ( s u l f i t e , e t c . ) , i s an a t t r a c t i v e and l o g i c a l model. 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