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The Influence of magnesium ions on the metabolism of phenylalanine by Pseudomonas Aeruginosa White, Mary Jacqueline 1949

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THE INFLUENCE OF MAGNESIUM IONS ON THE METABOLISM OF PHENYLALANINE BY PSEUDOMONAS AERUGINOSA • - by -Mary Jacqueline White A Thesis Submitted In P a r t i a l Fulfilment of the Reguirementa for the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the DEPARTMENT OF DAIRYING THE UNIVERSITY OF BRITISH COLUMBIA ABSTRACT The e f f e c t o f magnesium i o n s on the m e t a b o l i s m o f p h e n y l a l a n i n e by P a . a e r u g i n o s a haa been s t u d i e d . I n a g r o w i n g c u l t u r e , the e f f i c i e n c y o f u t i l i z a t i o n o f p h e n y l a l a n i n e by P a . a e r u g i n o s a was i n c r e a s e d by r a i s i n g t h e c o n c e n t r a t i o n o f M g C l 2 . 7 H 2 0 from 0.01% t o 2fo and d e c r e a s e d by r a i s i n g the s u l f a t e i o n p r o p o r t i o n a t e l y i n a g l y c e r o l - p h e n y l a l a n i n e - m i n e r a l medium. The optimum c o n c e n t r a t i o n o f magnesium f o r g r o w t h i n t h i s medium waa 1% M g C l 2 . 7 H 2 0 . The i n f l u e n c e o f v a r i o u s f a c t o r s o n t h e magnesium a c t i v a t i o n o f p h e n y l a l a n i n e o x i d a t i o n b y r e s t i n g c e l l s grown i n a m a g n e s i u m - d e f i c i e n t medium has been d e t e r m i n e d . The s t i m u l a t i o n by added magnesium i o n s was found t o be d e c r e a s e d by i n c r e a s i n g the c e l l c o n c e n t r a t i o n . O x i d a t i o n by c e l l s h a r v e s t e d f r o m a medium c o n t a i n i n g 0 .1% M g C l g . 7 H 2 0 was no t s t i m u l a t e d by added magnesium i o n s . The c o n c e n t r a t i o n o f added magnesium i o n a waa v a r i e d from 5 t o 5000 ppm. M g C l 2 . 7 H 2 0 and was found t o be s t i m u l a t i n g a t a l l c o n c e n t r a t i o n s t r i e d , a l t h o u g h 5000 ppm. was s l i g h t l y l e s s e f f e c t i v e . Magnesium as t h e s u l f a t e had a v a r i a b l e e f f e c t , u s u a l l y s t i m u l a t o r y b u t no t a a much as the c h l o r i d e . Manganous i o n s d i d n o t appear to r e p l a c e magnesium i o n s i n the s t i m u l a t i o n o f p h e n y l a l a n i n e o x i d a t i o n . The o x i d a t i o n o f 1 - p h e n y l a l a n i n e waa a l s o s t i m u l a t e d by added magnesium i o n s . The 1 i somer r e q u i r e d 6 moles o f oxygen p e r mole o f s u b s t r a t e whereas d l - p h e n y l a l a n i n e r e q u i r e d o n l y 4 m o l e s . The e f f e c t o f added magnesium i o n s on the o x i d a t i o n o f o t h e r amino a c i d a was s t u d i e d . L - t y r o a i n e and d l - v a l l n e were the o n l y amino a c i d a b e s i d e s d l - p h e n y l a l a n i n e whose o x i d a t i o n was a p p r e c i a b l y s t i m u l a t e d by added magnesium i o n s . A l t h o u g h the o x i d a t i o n o f d l - a l a n i n e was somewhat s t i m u l a t e d , t h e o x i d a t i o n o f d l - a e r i n e and l - g l u t a ; n i o a c i d was i n h i b i t e d by added magnesium i o n s . L - h i s t i d i n e was n o t o x i d i z e d e i t h e r i n t h e p r e s e n c e or ab sence o f magnesium i o n s . No s t r u c t u r a l r e l a t i o n s h i p b e t w e e n those ' compounds s t i m u l a t e d was e v i d e n t . The e f f e c t o f pH on t h e amount o f s t i m u l a t i o n by added magnesium i o n a was d e t e r m i n e d . O x i d a t i o n o f p h e n y l a l a n i n e i n c r e a s e d ove r a range o f pH 6 t o pH 8 bu t t he g r e a t e s t p e r c e n t s t i m u l a t i o n b y magnesium waa o b t a i n e d a t pH 6 . T h i a m i n and a m i x t u r e o f p y r i d o x i n e - p y r i d e x a m i n e were added t o d e t e r m i n e i f magnesium were s t i m u l a t i n g p h e n y l a l a n i n e o x i d a t i o n by a c t i n g as a n a c t i v a t o r o f t h e s e coenzyme p r e c u r s o r s . A l t h o u g the o x i d a t i o n o f p h e n y l a l a n i n e waa s t i m u l a t e d by b o t h t h i a m i n and p y r i d o x i n e - p y r i d o x a m i n e , t h e a c t i v a t i o n by magneaium i o n a waa no g r e a t e r i n t h e p r e s e n c e o f t h e s e a u b a t a n c e a . K e t o f i x a t i v e s were u sed t o d e t e r m i n e i f t h e s t i m u l a t e d r e a c t i o n o o o u r r e d b e f o r e the f o r m a t i o n o f a k e t o compound. -3-Araenite inhibited the oxidation completly both i n the presence and i n the absence of magnesium. Semicarbazide on the other hand, gave a 50% i n h b i t i o n , the i n h i b i t i o n being s l i g h t l y less i n the presence of magnesium ions. Cyanide did not i n h i b i t the oxidation at 1 x 10~2M or 1 x 10"3M. Since magnesium ions have been found essential to various phosphorylation mechanism, the p o s s i b i l i t y of phosphorylation of phenylalanine was suggested. Ho s i g n i f i c a n t phosphorus uptake waa found when phenylalanine waa oxidized nor did phorphorua appear to be essential to the oxidation of p phenylalanine or stimulationby magnesium ions. Deamination of the substrate was found to be s l i g h t l y i n h i b i t e d by magnesium. Since pH had been found to markedly influence the oxidation of phenylalanine and % stimulation by magnesium, i t was thought that by a determination of the R. Q. (ratio of C O 2 evolved to oxygen taken up), a difference inthe type of reaction ) occurring under these varying conditions might be found. The R. Q. i n the presence of magnesium ions at pH 6 and 7 and i n the presence or absence of magnesium ions at pH 8 wasfound to be greatly increased! (about 2 times). This would seem to indicate that under these conditions, reactions involving breaking the carbon chain are favored. An attempt was made to detect the possible intermediate compounds i n phenylalanine breakdown by oxidation i n the W arburg and by chromatography using the ninhydrin reaction. - 4 N e i t h e r t y r a m i n e , p h e n y l e t h y l amine n o r p h e n y l p y r u v a t e were o x i d i z e d a t any a p p r e c i a b l e r a t e i n the W a r b u r g . These compounds canno t be r u l e d out as p o s s i b l e i n t e r m e d i a t e s on t h i s b a s i s , however , s i n c e i m p e r m e a b i l i t y o f the c e l l may p r e v e n t t h e i r o x i d a t i o n . However , n e i t h e r t y r a m i n e , p h e n y l -e t h y l amine n o r t y r o s i n e c o u l d be d e t e c t e d by c h r o m a t o g r a p h y . I t was t h e r e f o r e c o n c l u d e d e i t h e r t h a t t h e s e compounds a r e n o t i n t e r m e d i a t e s i n t h e b reakdown o f p h e n y l a l a n i n e or t h a t more t h a n one method o f b reakdown i s o c c u r r i n g thus i n c r e a s i n g t h e d i f f i c u l t y o f d e t e r m i n i n g a n i n t e r m e d i a t e . : compound. A s e r i e s o f p h e n y l compounds waa t e s t e d to de te rmine i f magneaium were a l l o w i n g breakdown o f t h e p h e n y l r i n g . However , no o x i d a t i o n o f any o f the comnounda t e s t e d waa found to o c c u r . ACKNOWLEDGMENT The author wishes to thank Dr. J. J. R. Campbell for his encouragement and assistance during the course of this study and also Dr. B. A. Eagles for his hel p f u l suggestions. M. J. W. TABLE OF CONTENTS INTRODUCTION , 1 PART 1 THE INFLUENCE OF MAGNESIUM ON T PHENYLALANINE UTILIZATION AND GROWTH H i s t o r i c a l 3 Methods Ba c t e r i o l o g i c a l ......... - .8 Chemical , • • 9 Experimental and Discussion J.2 Conclusions...... 20 PART 2 FACTORS INFLUENCING THE STIMULATION OF PHENYLALANINE OXIDATION BY ADDED MAGNESIUM IONS H i s t o r i c a l 21 Methods Ba c t e r i o l o g i c a l •• 30 Experimental and Discussion 33 Conclusions • 56 PART 3 MECHANISM OF STIMULATION OF PHENYLALANINE OXIDATION BY ADDED MAGNESIUM IONS H i s t o r i c a l 57 Methods Ba c t e r i o l o g i c a l 67 Chemical 68 Experimental and Discussion 70 Conclusions 87 BIBLIOGRAPHY 89 Interest i n the effect of minerals on the amino acid metabolism of microorganisms was stimulated by Burton et a l ( 1 9 4 § ) who showed that the s a l t oonoentration i n a chemically defined medium greatly influenced the formation of the c h a r a c t e r i s t i c blue pigment pyocyanin from amino aoida by Ps. aeruginosa. This pigment i s formed as a by-product of nitrogen metabolism. It was therefore considered possible that minerals might be shown to influence the course of amino aold breakdown. Burton et a l (1948) had shown that adding phenylalanine to a mixture of amino acids allowing maximum pigment formation i n h i b i t e d the production of the pigment and that t h i s i n h i b i t i o n was removed by the addition of 8$ Mg304.7H20. Also, with phenylalanine ss the sole nitrogen source* no pigment was produoed i n a medium containing 0.01$ MgCl2.7H£0. Raising the concentration of t h i s mineral to 2$ however, allowed as much pigment formation as obtained with the optimum amino acid mixture used. These observations suggested that magnesium ions might play an important r o l e i n the metabolism of phenylalanine by Pa. aeruginosa. Phenylalanine was therefore chosen as the amino aoid to be studied. Aatudy of the e f f e c t of magnesium iona on phenylalanine u t i l i z a t i o n and b a c t e r i a l growth i n a medium containing this amino aoid as the sole nitrogeresource has been carried out. - a -The metabolism of phenylalanine by res t i n g c e l l s of Ps» aeruginosa In the presence and absence of added magnesium ions was also investigated. Part 1 of t h i s thesis i s concerned with the e f f e c t of varying the mineral constituents of the medium, especially magnesium, on the growth of the organism and the u t i l i z a t i o n of the amino acid with phenylalanine as the sole nitrogen source. The oxidation of phenylalanine by res t i n g c e l l s i n the Warburg apparatus was also found to be stimulated by added magnesium ions. The factors influencing t h i s stimulation are considered i n Part 2, In Part 3, some of the known mechanisms of amino acid breakdown were investigated using phenylalanine with and without added magnesium ions* In t h i s way, i t was hoped that an explanation of magnesium stimulation of phenylalanine oxidation might be found. PARS I THE INFLUENCE OF MAGNESIUM I0N3 ON PHENYLALANINE UTILIZATION AMI) GROWTH Magnesium i s a usual constituent of b a c t e r i o l o g i c a l growth media although studies conoerned with i t s e s s e n t i a l function have been r e l a t i v e l y slow i n development. Although as recently as 1942, Pandalai and Rao had reported that magnesium ions were not eaaehtial for growth °* Ps* aeruginosa • the combined use of improved cleaning methods for glassware, extraction techniques, r e p u r i f i e d chemicals, and double d i s t i l l e d water has shown magnesium to be an essential element for b a c t e r i a l growth and probably for a l l l i f e . Studies on the influence of magnesium on b a c t e r i a l growth have been carried out from many di f f e r e n t points of view. Using a medium oontaining p u r i f i e d chemicals i n r e d i s t i l l e d water. Young et a l (1944) found magnesium ions to be e s s e n t i a l for the normal growth of E. o o l l . Although f i v e micrograms per ten oo. was stated as the optimum con-centration (equivalent to 0.005$ MgCl2*7H20) of magnesium, no higher concentrations were t r i e d and thus i t i s possible that the peak of the growth - magnesium oonoentration curve was not reached. Brewer et a l (1946) stated that 0.000067 M added magnesium ions gave p r a c t i c a l l y optimum growth of B . anthraois as measured by spore oounts i n a chemloally -4-defined medium. Burton et a l (194'Q) demonstrated the neoeaaity of Mg304 for growth of Pa. aeruginosa. In fla s k s containing no added Mg304, no growth ooourred. In a leuoine - alanine medium, optimum growth was found at 0.5$ MgSQ^^HgO Tout pyooyanin formation was optimumat 3$. A diminution of growth occurred when added magnesium ions were omitted from the medium but s u f f i c i e n t magnesium was apparently present i n the medium to allow s l i g h t growth. Need of magnesium for adequate growth of the b a c t e r i a l o e l l i s harder to demonstrate with so;ne organisms than with others because of the necessity for a more oomplex growth medium. It was not u n t i l MoLeod and Snell (1947) added o i t r a t e to the medium that they were able to show a magnesium stimulation of the growth of c e r t a i n of the l a c t i c acid b acteria. Magnesium forms a complex with c i t r a t e and thus growth of the organism w i l l not occur i n a c i t r a t e medium unless excess magnesium ions are added. Five hundred micrograms of magnesium ion was found to give maximum growth of L. easel. The necessity of magnesium ions for normal c e l l d i v i s i o n was n i c e l y demonstrated by the data of Webb (1948). CI welohii cultures grown i n a peptone medium, shown to oontain l e s s than 0.00003$ magnesium ions, were found to contain large numbers of filamentous forms which had l o s t the Gram s t a i n oomplex (previously shown by Henry and Stacy (1943) to contain -5-magnaaium ribonuoleate). The magnesium content of the o e l l s waa moreover shown to be greatly influenoed by the concentrat-ion of magnesium i n the medium. Cells grown i n a low magnesium medium contained 2.5$ magnesium (of ash) whereas those grown i n a high magnesium medium contained twice t h i s amount. The l a g period i n the growth of Bact. i a c t l a aerogenes waa found to be influenced by the magnesium concentration i n a synthetic medium by Lodge and Hinahelwood (1939)* These workers found that an inoculum would not i n i t i a t e growth i n t h e i r medium i n the absence of magnesium. Calcium and magnesium ions were found to increase gelatinaae production by Proteua according to M e r r i l and Clark (1928). Haines (1933) diacovered that magnesium inoreaaed gelatinaae production by atimulating growth whereaa caloium increaaed gelatinaae production d i r e c t l y . Several atudiea have been carried out on the eff e c t of r e l a t i v e l y concentrated magnesium aolutions on microbiological growth. These have shown magnesium to be r e l a t i v e l y non-toxic u n t i l very high oonoentrations of the ion are used. Hotohkias (1923) showed 0.5M MgClg to be l i m i t i n g to the growth of E. o o l i i n a peptone medium; m u l t i p l i c a t i o n ocourred however i n 0.25M MgClg. Aooording to Mann (1932)t more than 0.0001 gm. mol. MgClg -6-per l i t r e waa needed, for the growth of A. niger. She toxic e f f e c t of higher concentrations of Mg Clg waa not antagonized by GSGlg and indeed O.lM CaCl 2 increased the toxic e f f e c t . McLeod and Smedley-HcLean (1938) found that 0.7% magnesium ions in h i b i t e d both carbohydrate and f a t synthesis i n yeast* Extensive work: on the effect of su r p r i s i n g l y high concentrations of magnesium on the growth of fungi and bacteria, has been carried out by Rabinovitz (1933). Some fungi were found to have a maximum tolerance of 40% Mg 304* The t o x i c i t y of higher concentrations was attributed to high osmotic pressure. Seven species of fungi, three of bacteia, four of blue-green algae a l l l i v e d i n media containing high magnesium, Also Rabinovitz showed that magnesium was more toxic unless balanced by calcium. The toxic action involved hindrance of oatalase action but stimulation of peroxidase* Winslow (1934) has emphasized a concept that i s often overlooked. A l l cations have a stimulatory e f f e c t at s u f f i c i e n t l y low concentrations and an i n h i b i t o r y e f f e c t at s u f f i c i e n t l y high concentrations. A compilation of the data of other workers showed the c r i t i c a l concentration of the various s a l t s to vary s l i g h t l y depending on the medium and also, i t i s probable, on the puri t y - o f the-medium used. For example, InjEi peptone solution, the stimulatory l e v e l of Mg C1 P was » - - - • - - .• • . . j . . . . . .. - _ . . . . . quoted as being 0.05 - 0.1 M but i n water, i t was stated as being 0.02 - 0.08M. 0.5M MgCl 2 had been found to be highly toxio* -7-Ihe survival of bacteria i n solutions of MgClg was ompared with a s a l t - f r e e contBol;119$ survived at 0.01M concentration of MgClg but only 66$ at 0.1M MgClg* Magnesium ohloride was found to be about eight times as powerful as sodium ohloride i n the stimulatory and i n h i b i t o r y zones* Aooording to Winslow. permeability was inoreased by aodium and other univalent ions and decreased by calcium and other divalent ions* •8 METHODS Baoterlologloal: Pa. aeruginosa A.T.O. 9027 waa used i n a l l experiments. The medium uaed f o r atooka and transfer medium oonaiated of: gl y c e r o l 1$ peptone (Bacto) 1 tap water pH 7.8 Twenty-four hour agar stab cultures were refrigerated as atoots. An inoculum from the atook culture was transferred d a i l y at leaat three times before use. The inoculum for a l l experiments oonaiated of one drop of a one to twenty-d i l u t i o n of a 24 hour culture i n r e d i s t i l l e d water. A l l glassware was cleaned by soaking overnight i n a 10$ n i t r i o aoid solution followed by rin a i n g i n tap water, d i s t i l l e d water, and f i n a l l y i n r e d i s t i l l e d water. The fla s k s were autoclaved with r e d i s t i l l e d water before use. A l l solutions were made upjfrom reagent grade chemicals uaing water r e d i s t i l l e d through glass. Media were dispensed i n 125 Ml. or 250 ml. erlynmeyer fla s k s with beakera aa tops. The "low s a l t s " basal medium of Burton et a l (1947) waa used. Thia oonaiated of: glycerol 1$ amino aoid 1 K0HPO4 0.02 F8SO4?7Hg0 0.0004 MgS04.7H20 0.01 -9-For the "high a a l t a " medium, the three s a l t s were inoreased as follows: In the experiments i n which either magnesium or sulfate ion were varied separately, the other ion was used i n the basal (that i s , 0.006% Ha 230 4 or 0.01% MgCL 2.7H 20)• In a l l Cases}, the basal medium, containing the g l y c e r o l , amino acid and a l l of the s a l t s except those to be varied, was made up i n a concentrated form. After being dispensed into the flas k s , the mineral solutions containing the s a l t s to be varied were added, the pH of the medium was adjusted to 7.2, and water was added to make a f i n a l volume of l O c c or 25 oo. Incubation was at 3 0 ° C , for 4 days, Uninoculated flasks served as contols. At the end of the incubation period, o e l l s were oentrifuged down and the supernatant was analyzed for r e s i d u a l phenylalanine. B a c t e r i a l growth was determined 1 as o e l l nitrogen. She o e l l s were analyzed a f t e r being washed twice i n phyaiologioal s a l i n e . Chemical: Magnesium chloride solutions used i n these experiments were standardized against AgNOg using potassium chromate as indicato r ; this was necessary beoause of the extremely hygroscopic nature of the s a l t . 0.04% 0.001 2.0 medium i n the concentration as contained i n 0. 01% Mg304.7H20. -10-Phenylalanine waa determined by the method of Block and B o i l i n g (1940). Sampled containing not more than 2 mg. of phenylalanine were dried on a steam bath. Following n i t r a t i o n with a potassium n i t r a t e - s u l f u r i c aoid mixture, the hydroxylamine derivative was formed; the purple ammonium aalt was then formed by the addition of conoentrated ammonium hydroxide and the coloured solutions were read i n the Fiacher eleotrophotometer using the f i l t e r g iving maximum absorption at 525 Mu. A standard curve was run for each set of determinations and also a protein blank (sample with no added hydroxylamine). Results were expressed aa mgm. of phenylalanine used per oo. of oulture medium. C e i l nitrogen was determined by the micro-Kjaldahl teohnlque. Cells were washed twice with ph y s i o l o g i c a l aaline and were reauspended i n three oo. of d i s t i l l e d water. One cc. aliquota were used for anal y s i s . The samples were digested over micro-burners using one co. concentrated Eg3Q4 and a few dropa of hydrogen peroxide aa oxidizing catalyat. Digestion waa carried on u n t i l a clear aolution which did not darken on aubaequent heating waa obtained. The digested aamplea were steam d i s t i l l e d into N/lOO H2SO4 and the receiving flaska were back-titrated with U/lOO NaOH. Reaulta were expreaaed aa mgm. of o e l l nitrogen per oo. Magneaium waa determined uaing a modification of the molybdivanadate method of Simonaen et a l (1947). Samples containing not more than 100 micrograms of magneaium were - l i -t r e s ted with ammonium hydroxide and potas alum phosphate to preci p i t a t e the magnesium as magnesium ammonium phosphate. She precipitate was oentrifuged down and was washed with an alcohol - ammonium hydroxide mixture. The precipitates were dried and the phosphate was determined by the colour reaction with molybdate and amino-naphtho sulfonic acid according to the method of King (1932). -12-EZPERIMMTAL AND DISCUSSION Each of the s a l t s i n the ;t>asal medium was used separately i n an increased concentration to determine i t s effect on amino aoid u t i l i z a t i o n and growth (Table 1)* Magnesium and sulfate ions were inoreased separately since Burton et a l (1948) had shown that, whereas they both increased pigment production i n an inoreased concentration, magnesium increased growth but suifatd did not. Duplicate flasks were used. From Sable 1, i t i s apparent that, although both growth and amino aoid u t i l i z a t i o n are greatly inoreased i n a "high s a l t " medium, the r a t i o of amino acid used to b a c t e r i a l nitrogen produoed i a r e l a t i v e l y unchanged. Two,percent Mg304.7H20 appears to be s l i g h t l y t o x i c to growth but the amino aoid u t i l i z a t i o n waa i f anything s l i g h t l y increased over that occurring In the "low s a l t s " medium. The most s t r i k i n g effeot was found when magnesium and i sulfate ions were increased separately. I f the r a t i o of amino acid used to c e l l nitrogen produced i s considered as an ef f i c i e n c y r a t i o , i t i a apparent that a high concentration of magnesium ion greatly Increased the e f f i c i e n c y of u t i l i z a t i o n of the amino aoid whereas increasing the aulfatejion proportionately on a molar baa i s , greatly decreases the effielenoy. The nitrogen metaboliam e f f i c i e n c y r a t i o as -13 Table 1 Influence of s a l t s on growth and u t i l i z a t i o n of •phenylalanine. BACT. H. (mg./lO op. P.A. P.A. used MEDIUM - • - • • • used mg./oc. Baot. H. lype C2HP04 Fe30 4. . 7H20 jo M g S 0 4 „ Low s a l t s 0.02 0.0604 0.01 0.91 4.0 4.2 b.igh ** 0.04 0.0010 2.0 1.6 7.0 4.3 ligh MgSO iO.02 0.0004 2.0 0.73 4.3 5.9 " K2HPO4 0.04 0.0004 0.01 0.55 6.3 11.0 " FeS0 4 0.02 0.0010 0.01 0.42 4.4 11.0 " Mg ion 0.02 0.0004 # 1 # 3 0.69 2.6 3.7 " S0 4 " 0.08 0.0004 #2 0*16 2.5 16.0 M g C l o . 7 H g 0 E.0% * Na 2S0 4 * 0.006% M g C l 2 . 7 H 2 0 0.01$ Ha2S04 1.2 % # single flask: determinations. -14-oaloulated from the data of Kazal (1947) for Ps. fiugresoens grown for four daya on a glutamic aoid - inorganio s a l t s medium and Sartory et a l (1946) for Staph, sp. grown on peptone broth was found to be about f i v e . Our r a t i o s were found to vary with the s a l t concentrations within a range of 3.7-16. I t i s considered possible that the apparent greater e f f i c i e n c y of u t i l i z a t i o n of the amino acid i n a high magnesium medium as indicated by a lower r a t i o , might be due to a greater s e n s i t i v i t y of growth of the organism than breakdown of the amino acid to the concentration of magnesium ion. So determine more exactly the effect of magnesium concentration on growth and phenylalanine u t i l i z a t i o n , an experiment was set up i n which the magnesium ion concentration was varied through a range of 0.01% MgClg^HgO to 2% MgCl2.7H2& leaving the sulfate ion constant at 0.006% (equivalent to 0.01% Mg304.7H20) as i n the low s a l t mixture (Table 2). She optimum concentration for growth apparently i s at 1% and f a l l s o f f s l i g h t l y at 2%. On the other hand, amino acid u t i l i z a t i o n i s only s l i g h t l y affected by magnesium ion concentration and no rel a t i o n s h i p between concentration of magnesium and u t i l i z a t i o n of the amino aoid i s apparent from th i s data. These magnesium ion concentrations are much higher than has been ci t e d i n the l i t e r a t u r e as providing optimum growth for microorganisms. -15-Table 2 Effeot of MgClg.7HgO concentration on  growth and phenylalanine u t i l i z a t i o n COHC. Ug0l2.7H20 Bact, U mg./lO co. P.A. used mg./oo. P.A. used Bact. E 0,01' 0.08 5.3 66.0 0.06 0,26 5.1 19,0 0.1 0.45 4.0* 9.0 0.3 0.60 3.8 6.3 0.5 0.64^ 5.8 # 9.0 1.0 0.82 4.1 5.0 2.0 0.64 325 5.5 # single f l a s k determinations. - 1 6 -A s i m i l a r experiment was carried out to show the effect of varying sulfate ion independently on growth and amino acid breakdown* (Table 3)* The optimum for growth of the organism appeared to be at 0*6% NagSO^ although there was actually very l i t t l e difference between th i s concentration and the lowest concentration 0.06% RagSO^ Definite i n h i b i t i o n of growth was apparent at 1% Na2S04 with even greater i n h i b i t i o n at 1.2%. The greatest breakdown of phenylalanine occurred at 0*06% NagS04 and t h i s was progressively decreased as the sulfate ion concentration was raised* From these two experiments i t can be seen that r a i s i n g the concentration of magnesium ion as MgS04 involves introducing another ion which may partly n u l l i f y the effect of the magnesium ion. Because of the high concentration of magnesium apparently needed for optimum growth, i t waa of interest to determine i f any magnesium had been taken up from the medium at the end of four days growth* Table 4* No s i g n i f i c a n t uptake of the mineral was found to havo occurred. The reason for the necessity of having such a high concentration of magnesium ions i n a medium f o r the growth of Ps. aeruginosa as found by Burton et a l (1948) and also i n t h i s study, i s obsoure. Since a medium as chemically defined as can be obtained by the use of r e d i s t i l l e d water, chemically clean glassware and high-grade ohemioals has - 1 7 -Table 3 Effect of sulphate Ion concentration on  growth and phenylalanine u t i l i z a t i o n * Na 230 4 . . MgC1^.7H2P Cells Turbidity# Phenylalanine used - mg./oo. 0.Q6 0.1 55 1.4 0.6 0.1 61 1.2 1.0 0.1 22 0.59 1.2 0.1 14 0.59 # Reading on log. soale of Fischer Eleotrophotometer, -18-Table 4 Magneaium uptake from medium at end of a four day growth period* MgCl2.7H2G # i n medium Sample MgCl 2.7H 20 at s t a r t miorogra >ms Sample MgCl 2.7H 20 >:• at f i n i s h micrograms 0.1 100 100 0.5 500 450 1.0 1000 1050 2.5 2600 2500 - 1 9 -been employed throughout, the concentrations of minerals which must be added w i l l probably be greater than has been found by certain other workers. This factor alone however does not explain the great discrepancy between the optimum concentration found i n these experiments and those usually oited i n the l i t e r a t u r e . -20-CONCLUSIOffS Growth and amino aoid u t i l i z a t i o n by Pa, aeruginosa i n a chemically defined medium containing glycerol and phenylalanine haa been shown to be influenced by varying the mineral constituents. Increased growth and e f f i c i e n c y of u t i l i z a t i o n of phenylalanine has been obtained by r a i s i n g the magnesium concentration. One percent added MgClg was found to be the optimum concentration for growth i n a g l y c e r o l -phenylalanine medium. Eaising the sulfate ion proportionately was found to depress growth and e f f i c i e n c y of u t i l i z a t i o n . Uo s i g n i f i c a n t amount of magnesium was found to have been removed from the medium by the c e l l s at the end of fouf~ days growth. -21 PARI S FACTORS INFLUENCING (THE STIMULATION OF PHENYLALANINE OXIDATION BY ADDED MAGNESIUM 10N3 The d i f f i c u l t i e s involved i n demonstrating the s p e c i f i c i t y of activator-metabolite relationships using growing oultaxes may i n part be overoome by the use of the Warburg technique with r e s t i n g c e l l s . The possible number of reactions occurring at one time i s greatly decreased by th i s method since only one substrate i s present and synthetic processes are at a minimum. Preliminary experiments with r e s t i n g c e l l s of Ps. aeruginosa indioated that added magnesium ions stimulated the oxidation of d l phenylalanine. In an ef f o r t to determine the mechanism of the stimulation, further study was made of the conditions influencing this stimulation* Amino acid metabolism Extensive work on the breakdown of amino acids by bacteria has been carried out by Gale (1940). I t was shown that the method of primary attack of the amino acid molecule by r e s t i n g c e l l s of bacteria i s greatly influenced by the pH of both the growth medium and the c e l l suspending medium. At an acid pH, the amino acid i s f i r s t deoarboxylated with -22-the formation of a primary amine. At an a l k a l i n e pH, deam-ina t i o n predominates with the formation of a keto aoid i f the reaction i s oxidative. Gale has suggested that, since these reactions tend to neutralize the medium, they may be considered as protective mechanisms and may thua not have the metabolio significance often ascribed to them. Indeed, i t has been stated that at n e u t r a l i t y , theae reactions are at a minimum* Ever alnce Gale (1940) suggeated the p o s s i b i l i t y of the necessity of a coenzyme for deaminases - c a l l e d "©©deaminases" - reports of the discovery of atimulators of amino aoid metabolism have appeared i n the l i t e r a t u r e * L i c h atein and Umbreit (1947) reported b i o t i n a c t i v a t i o n of threonine, aapartio aoid and serine deaminases* In a study of the breakdown of several amino acids by CI w e l o h l l . Woods and Trim (1942) obtained results which they interpreted aa i n d i c a t i n g the neceasity of a coenzyme factor for serine breakdown. The concentration of the o e l l auapenaion markedly affooted the oxidation. The higher rate of oxidation could be obtained by adding boiled organiama.but not by adding a small part of the o r i g i n a l growth medium. That the atimulation may have been pa r t l y inorganic i s suggeated by the experimenta of Binkley (1943) who found serine breakdown by an enzyme preparation from E. o o l l to be atimulated by 0.001M zino, manganese, or magnesium* - 2 3 -The pyridoxins - pyridoxamine - pyridoxal group of growth factors have been shown to function i n several oapacities i n amino aoid metabolism. Gunsalus et a l (1944) showed the tyrosine decarboxylase system of 3o. f a e c a l i s to be stimulated by pyridoxal. The reaction pyridoxal + ammonia pyridoxamine was found to be s i g n i f i c a n t i n transamination reactions by Snell (1945). Wood et a l (1947) showed pyridoxal phosphate to be the coenzyme of the tryptophanase system of E. c o l l (oodecarboxylase). i n h i b i t o r s have been used f a i r l y extensively to determine the course of amino acid breakdown by both tissues and b a c t e r i a l enzyme systems. By the use of O.OOIM arsenite, Krebs (1933) obtained the accumulation of keto acids from the oxidative deamination of amino acids by kidney s l i c e s * The amount of ammonia formed agreed olosely with the amount of keto aoid obtained by the formation of the 2:4 dinitrophenylhydrazine derivative. Krebs (1935) suoeeded i n d i f f e r e n t i a t i n g between d- and 1 amino acid deaminases of l i v e r and kidney tissue by the use of o c t y l alcohol and cyanide i n h i b i t o r s . Ihe oxidative deamination of the natural isomers was i n h i b i t e d by o c t y l alcohol, cyanide, drying or d i l u t i o n of the tissues whereas the oxidation of the unnatural isomers was unaffected by these factors* Tyrosine oxidation i n l i v e r was found to be i n h i b i t e d with KON (0.005M) and phenylalanine oxidation i n kidney to be unaffected acoording to the work of Bernheim and Bernheim -24-Both oxidations wore stated to be unaffeoted by s a l t concentrations and f l u o r i d e . A kidney tissue enzyme preparation which p r e f e r e n t i a l l y oxidized the unnatural isomers of alanine, phenylalanine, valine and leucine was found by Bernheim and Bernheim (1935) to be unaffected by KCN. S i m i l a r l y * Lang (1942) found 1 phenylalanine oxidase to be unaffected by HON. Edlbacher and Grauer (1944) were able to show a selective action of various i n h i b i t o r s on the oxidative deamination of 1 phenylalanine and 1 glutamic acid by guinea pi g kidney s l i o e s . The decomposition of 1 phenylalanine was i n h i b i t e d by 0.001M ECS, O.IM NaP, 0.05M malonio acid and 0.001M iodoaoetio aoid, whereas 1 glutamic acid oxidation was i n h i b i t e d only by the cyanide. 0.001M arsenious aoid i n general i n h i b i t e d not at a l l . The oxidation of several amino acids by " r e s t i n g " B a c i l l u s proteua (sic) was i n h i b i t e d by 0.00511 ZCN acoording to Bernheim et a l (1935). Webster and Bernheim (1936) found that 0.005M ECU also i n h i b i t e d the oxidation of amino acids by Ps. aeruginosa. Sodium fl u o r i d e and urethane were also i n h i b i t o r y . Binkley (1943) i n a study of dialysed enzyme preparations from E. o o l i found 0.001M fluo r i d e to be i n -h i b i t o r y . A c t i v i t y of the dialysed prepartion was restored by adding 0.001M zinc, manganese or magnesium and i t was only when the preparation was reactivated by zinc that 0.001M HCU was found to inactivate the enzyme oxid i z i n g serine. The action of i n h i b i t o r s on b a c t e r i a l amino aoid decarboxylases -25-waa studied by Taylor and Gale (1945). The u t i l i z a t i o n of the d- or unnatural isomers of the amino acida has been studied and has been found to vary from species to species. The u n a v a i l a b i l i t y of the d form of alanine for dogs, rats' and rabbits i s indicated by the work of Abderhalden (1935) who found d alanine i n the urine of animals fed d l alanine. The pure d aoid fed to the animals apparently waa t o x i c . Although i t has generally been conaidered that humans are able to u t i l i z e both forma, Albaneae (1942) found great differences between lndividuala i n the amount of d isomer uaed probably due to the preaence of varying amounts of d amino acid oxidase* Grauc.' (1947) found both isomers of phenylalanine to be used by chicks aa In the rat and the mouse. Webster and Bernheim (1936) divided the amino acids oxidized by Pa. aeruginosa into three groups on the basia of o p t i c a l a p e c i f i c i t y : 1. natural iaomera only attacked - leucine, iaoleuoine, h i s t i d i n e 2. non-natural iaomera increase oxygen uptake but no deamination - phenylalanine, valine 3* both isomers attacked - alanine, serine tyrosine, p r o l i n e . The d forms of leucine and valine were found to be i n h i b i t o r y to L. arablnaoaua by F l i n g and Fox (1945). Kobayaahi et a l (1948) i n a atudy of amino acid r e l a t i o n -ahipa, found the d forma to i n h i b i t at much leaser concen-trations than the 1 forma. I t waa suggested that the d l amino aoida may not have the same resultant action as the aum of -26-of the d and the 1 forma and thereforek i n a study of t h e i r r e l a t i v e a c t i v i t i e s , the pure Isomers should be compared rather than the d l mixture.with one of the o p t i c a l l y active isomers. Factors influencing magnesium stimulation of enzyme systems Various workers have reported magnesium stimulation of enzyme systems to be i n h i b i t e d by other metallic ions. Ohlmeyer (1941) reported a c t i v a t i o n of enolase to be in h i b i t e d by calcium and strontium ions. Magnesium was found by Bielschowsky et a l (1946) to decrease the i n t e n s i t y of oardio-vascular effects of certain purine derivatives probably by diminishing the rate of deamination. The action of magnesium i n this case also waa found to be reversed by calcium. According to Nagamma and Narayana (1948), stimulation of the pyrophosphatases of erythrocytes and yeast by magnesium ions was also antagonized by calcium. As found by the work of Stoner and Green (1945), magnesium added as the sulfate may give i r r e g u l a r r e s u l t s . Aoco.rdl.ng to these workers, magnesium as the chloride i n h i b i t e d the dephosphorylation of adenosine triphosphate but as the sulfate had a variable e f f e c t , c h i e f l y acceleration. Zimmerman (1947) ha a atated that magnesium as the chloride i s more available to plants than as the s u l f a t e . - 2 7 -Othar iona, eapeoially manganaae, nave frequently been reported to replaoe magneaium aa stimulators of enzyme systems. Lobman and Schuster (1937) found magnesium or manganese ions to stimulate the decarboxylation of pyruvate by washed yeaat auapenaions. From t h e i r r e s u l t s , i t appears that ten micrograms of manganaae gave aa much stimulation of the reaction aa 100 micrograma of magneaium. I n t e s t i n a l dipeptidaae was found by Berger and Johnson (1939) to be activated by either magneaium or manganese. Manganese iona were found by Adler et a l (1939) to be more active than magneaium i n the atimulation of i s o c i t r i c dehydrogenase aotion i n animal tiasuea. Erampitz and Werkman (1941) showed magnesium or manganese to be needed for decarboxylation of oxalaoetate by a decarboxylase proportion from M. lyaodeltfcloua. Manganese waa found to be moat aotive i n the decarboxylation of pyruvate by an enzyme preparation from Proteus by Stumpf (1 (1945). I t was stated however, that magnesium probably oocurs i n the natural system. On the other hand, ELeinzeller (1940) found that manganese would not replace magnesium i n atimulation of the oxidation of fumarate by pigeon breast musole i n phosphate buffer. That these two ions may not always be interchangeable i a further shown by E i l l a o n et a l (1942) who regenerated a f l u o r i d e -poisoned aloohol fermentation by the addition of MnClg but not by MgClg. In t h i s paper however,they atate that with -28-Azot, phrooooooum, B, radlobaeter, and B. prodlgioaum, the magnesium of the medium may be replaced by manganese* Trigga (1949) found however, that with a species of Betacoeoua, no growth was obtained i n the absence of magnesium i n a medium containing manganese. Manganese was found to be stimulatory to growth* Warburg and C h r i s t i a n (1941) found that either magnesium, manganese, or zino would activate c r y s t a l l i z e d enolaae. Manganese, calcium or magnesium reactivated dialyzed phosphatase enzyme preparations aa shown by Roohe et a l (1943). Zino did not reinforce phosphatase a c t i v i t y as had been reported by another worker. Binkley (1943) demonstrated re a c t i v a t i o n of dialyzed serine deaminase and cysteine desulfurase a c t i v i t y by 0.001M magnesium, manganese or zino. Either calcium, magnesium, manganese or barium antagonized the growth i n h i b i t i o n of E. o o l i by atabrine as found by Silverman (1948). Modification of an ion stimulation by the pH of the reaction i s Indicated by the data of Nguyen-Van (1942) who found that at pH 6-6, the a o t l v l t y of emulsin of almonds was inhib i t e d by magnesium or calcium ions but at pH 3-6, the enzyme was strongly activated by magnesium and calcium ions. This data was interpreted aa showing the preaenoe of two separate enzymes. Part of the differences i n stimulation at various pH's may be explained by the varying s t a b i l i t y of enzyme-mineral complexes at d i f f e r e n t pH's. -29 Kubowitz and Luttgena (1941) found no loss of a o t i v i t y In thiamin-pyrophosphate-magnesium-protein complex when dialyzed at pH 5. At pH 8, cleavage of the complex occurred with consequent loss of aotivators by d i a l y a i a , Che a c t i v i t y could be restored 85% by the addition of magnesium and thiamin pyrophosphate. Quastel and Webley (1942) found the % a c t i v a t i o n of depleted o e l l s to be greater the lower the pH. •30-MBTH0D3 Bacterlologioal; The organism used for Itheae studies was Pa. aeruginosa A.T. C. 9027, The stock was kept i n the r e f r i g e r a t o r on gly c e r o l peptone agar (glyeerol 1%, peptone 1%, pH 7,8). The culture was transferred from day to day i n glycerol peptone broth. A new culture was taken from stock every two weeks. I t was necessary that the medium f i n a l l y adopted should allow the growth of s u f f i c i e n t numbers of magnesium-deficient c e l l s to attack phenylalanine at a f a i r l y rapid rate. Ho sugar was used i n the medium since Gale and Stephenson (1938) and Bpps and Gale (1942) had found sugar to i n h i b i t the form-ation of amino aoid s p l i t t i n g enzymes. During the l a s t part of these experiments, i t was decided to add c i t r a t e to the growth medium since many workers (for example, Feldberg and Helb (1947), Lanaing et a l (1942), Monnier et a l (1947), had shown magnesium to form oomplexes with c i t r a t e . The stimulation with added magnesium would then become more apparent. Since, aooording to Gale (1940), enzymes s p l i t t i n g amino acids have been found to be adaptive, an acid hydrolysis of casein, Bacto easaminoacids was used as the sole nitrogen and energy source inthe medium. The medium f i n a l l y adopted consisted of caaamino acids 0.5%, K 2 H P O 4 0.078%, FeS04.7H20 -31-0.001$, aodium c i t r a t e 0.2$, pH 7.2-7.4. The medium waa made up with d i s t i l l e d water and was dispensed i n 100 co, lo t s into Roux flasks which had previously been rinsed several times i n d i s t i l l e d water. A 1$ inoculum of a 10-24 hour culture waa used and incubation was at 30^0. It was found by preliminary experiment that c e l l s could be used af t e r from 12 to 24 houra incubation. However, a f t e r 24 hours, the growth flasks became very gummy and removal of thecells by centrifugation waa p r a c t i c a l l y impossible. The c e l l s were collected by centrifugation i n a Serval angle oentrifuge using p l a s t i c cups which had been rinsed several times i n r e d i s t i l l e d water. They were washed twice i n physiological saline using glass cups which had been chemically oleaned. A f i n a l suspension i n saline which had a concentration of 33 to 40 times that occurring i n the growth medium was prepared. The Warburg technique (Umbreit, 1945) was used i n the following experiments. The temperature of the bath was at 31°C. A l l substrates were added from the sidearm a f t e r temperature e q u i l i b r a t i o n of the f l a s k s . Substrates were neutralized prior to use and were used i n such a concentration ,,that complete oxidation would require 400 u l . of oxygen. I t waa necessary to use rigorous cleaning methods to ensure as complete removal of contaminating ions as possible. A l l glassware was soaked i n aqua regie and was then rinsed i n - 3 2 -tap water, then four times i n water r e d i s t i l l e d through glass. A l l solutions were made up i n r e d i s t i l l e d water. Oxygen uptake was followed for a period of from two to four hours. Sinoe i t was considered possible that contaminating organisms might affect the results i n a four hour run, Gram s t a i n from each cup were made at the end of the run for s i x consecutive runs. Bo contaminants were found. The endogenous value was subtracted from the value for the substrate f l a s k i n a l l experiments except where otherwise indicated. Suitable endogenous oontrols were used. 33-EXPEKIMENTAL AND DISCUSSION Ca l l s : I t was of interest to determine the effect of o e l l concentration on the stimulation of phenylalanine oxidation by added magnesium ions. In th i s experiments, 0.05% MgCl2.7HgO was used i n the cups. The concentration of c e l l s used were 20, 35 and 50 times growth concentration. I t i s evident from Table 1 that increasing the concentration of the o e l l s decreases the magnesium stimulation. Since i t i s realized that the c e l l s , though they be magnesium-deficient, must c contain a c e r t a i n amount of the mineral, i t i s probable that the decrease of stimulation i s caused merely by the addition of greater quantities of the stimulating ions to the flasks through the medium of the c e l l s . I t was decided therefore to use 40x o e l l s i n future experiments to ensure as small an i n i t i a l magnesium content of the flasks as possible commensurate with s u f f i c i e n t l y active breakdown of the amino acid. In order to show the extent to which the o e l l s used were magnesium-deficient and to discover whether they could absorb magnesium into the o e l l from the medium i n s u f f i c i e n t quantity to allow an optimum concentration of magnesium i n the cups, o e l l s grown i n the magnesium-deficient medium and oe l l s grown i n the usual medium with 0.1% MgCl p.7H g0 were -34 Table 1 Bffeot of o e i l concentration' on $ atimulation by added magneaium ions u l * oxygen uptake at end of two hours C e l l * cono. (times growth . cono*) No added Mg 6G00 ppm. MgCl 2.7H 20 $ atimulation 20 35 23 60 160 61 133 119 50 113 149 32 In cups: M/15 phosphate buffer 1*6 oo* pH 7.2 C e l l suapension (saline) 0.6 " Phenylalanine - .29 Mg. /oup 0*2 " (sidearm) KOH - 20$ (centre well) 0.16" Mg012.7H20 - 50,000 ppm. 0.3 " H20 up to 3*15" •35-oompared i n the Warburg aa to oxidation of phenylalanine and atimulation of the oxidation by 5 ppm. added MgCl2.7HgO. The magnesium chloride waa added to the medium a a e p t i c a l l y a f t e r s t e r i l i z a t i o n of the medium i n order to avoid the formation of a magnesium phosphate p r e c i p i t a t e . In t h i s case, the c e l l s were washed three times i n saline to ensure more complete removal of the medium from the o e l l s . Table 2. As seen from the table, oxidation by c e l l s grown i n 0.1% MgCl 2.7H 20 i s not stimulated by the addition of magnesium ions. F i g . 1 shows t y p i o a l curves obtained showing magnesium atimulation of dl-phenylalanine oxidation. In order to allow the reaction to run to completion, oxidation waa allowed to proceed' for four houra. I t would appear that the atimulation involvea a "speeding-up" of the reaction and that the same type of breakdown i s ocourring i n both cases since the curve flattened out i n the same plaoe. Four moles of oxygen were taken up per mole of dl-phenylalanine* Magnesium concentration: To determine i f the concentration of added magnesium ions influenced the amount of stimulation of the oxidation, 5, 50, 500 and 5000 ppm. ( f i n a l concentration) MgCl2.7H20 were added to the oups. F i g . 2. I n general, the greatest °/o stimulation occurred a f t e r 100 to 120 mlns. Over the range 5 - 5000 ppm. Mg01g.7H20, muoh the same % a c t i v a t i o n was obtained. Five thousand ppm. MgCl2*7H20 -36Q Table 2 Effect of magnesium content of medium on  $ atimulation of phenylalanine oxidation oxygen uptake at end of 160 mina. - u l . Cells Mg-deficient Mg-grown i n cups No added Mg 5 ppm. MgCl2.7H20 No added Mg Mg e?I^ H2° time -mina. 40 59 80 57 61 80 97 135 90 90 100 116 170 116 120 120 139 222 148 151 140 166 236 185 185 160 188 250 204 203 Eleotrophc reading oi t . 76.5$ 85$ -37-280 Figure I: Effedt of added magneaium iona on oxidation of dl-phenylalanine ...... plug 5 ppm. M g C l 2 . 7 H 2 0 ( f i n a l concentration) 50 " 500 " 5000 " Figure 2: Effect of mineral concentration on atimulation of dl-phenylalanine oxidation by Mg iona. -39-appeared to be s l i g h t l y less e f f e c t i v e . The r e l a t i v e low t o x i c i t y of the magnesium ion i a apparent from these experiments. 3D iaomer: Since d iaomera of amino aoida have been reported to be to x i c i n aome caaea, i t waa decided to compare oxidation of the I iaomer with that of the d l aoid. Fig. 3 ahowa the 1 iaomer to be atimulated alao although oxidation waa greater - 6 molea oxygen per mole of aubatrate aa compared to 4 molea oxygen per mole for the d l iaomer. From these data, i f the oxidation of the d l acid iajconaidered aa the reaultant of the oxidation of the two iaomera, i t appears that the oxidation of the d iaomer atopa when 2 molea of oxygen have been uaed. The stimulation of phenylalanine oxidation by magnesium iona acoording to t h i a experiment has no iaomer s p e c i f i c i t y . The unnatural isomer doea not appear to be i n h i b i t o r y . Substrate concentration: Ittwaa decided to determine i f the atimulation waa related to the n e u t r a l i z a t i o n of c e r t a i n toxic compounds by magneaium iona aa reported by Maaaart (1947) for dyea and Bodansfcy (1949) for amino acids. The concentration of the substrate was increased up to four timea that o r d i n a r i l y used. Stimulation was obtained i n a l l cases, the greatest $ stimulation occurring with three Figure 3: Stimulation of oxidation of dl-phenylalanine and 1-phenylalanine by 5 ppm. added MgClg.7HgO -41-timea the usual concentration of substrate. No t o x i c i t y , was apparent at the higher concentrations. F i g . 4. Other amino acids; By test i n g the stimulatory influence of magnesium ions on other amino acids, i t was hoped that oertain s t r u c t u r a l relationships of those stimulated might be revealed. The substrates used were l-ibyrosine, 1-leucine, dl-alanine, d l - v a l i n e , 1 - h i s t i d i n e , dl-serine and 1-glutamio aoid. Figs. 5.6,7. Of these, dl-valine and 1-tyrosine were the only amino acids besides dl-phenylalanine to be stimulated. Dl-alanine was very s l i g h t l y stimulated. I - h i s t i d i n e was not oxidized at any appreciable rate either with or without added magnesium ions. Table 3, Added magnesium ions appeared to be s l i g h t l y i n h i b i t o r y to the oxidation of dl-serine and 1-glutamic aoid. The stimulation of 1-tyrosine suggests that t h i s compound may be an intermediate i n phenylalanine oxidation by Ps. aeruginosa as found by Beerstecher and Shive (1947) for E. c o l i . Four oxygen moles per mole of substrate were taken up by the oxidation of 1 tyrosine as compared with 6 moles of oxygen per mole for 1 phenylalanine. The reason for stimulation of dl - v a l i n e , and dl-alanine, amino acids less oomplex i n struoture than for example, 1-leuoine, which was not stimulated, i s obscure. Figure 4: Oxidation of increaalngdl-phenylalanlne ~ 1 " •"' "c'ono'entratlon witn no added magneaium Tone• # atimulation by added magneaium iona p 6 ppm. MgCl2.7Hg0 Mina • 1.8 urn. 3.6 urn. 5.4 urn. 7.2 urn. 20 63 157 " 40 10 56 120 105 70 37 38 67 79 100 39 42 52 70 120 30 "36 71 72 140 14 54 86 73 F i g u r e 5 : Magnesium s t i m u l a t i o n o f 1 - t y r o s i n e and d l - a l a n i n e o x i d a t i o n . 1-leucine " plus Mg T E X i . . . ^ r -Figure 6: Magneaium a t i mulation of £•' r: and 1-leucine oxidation (andogenoua not subtracted.) - 4 s -F i g u r e 6: S t i m u l a t i o n o f d l - v a l i n e o x i d a t i o n by added Mg i o n a . -46-Table 3 Effect o f added MgCl g.7H 20 (5 ppm.Vgon oxidation  o f d l - a e r i n e , i-glutamlo acid and j - h l a t i d l n e ul * , feXRgQfl uptake 1 MSI . dl-aerine l-gjutamic 1-hiatidine 40 No Mg 125 Mg 128 No Mg 70 Mg 60 No Mg 2 Mg 0 60 197 203 117 104 11 7 80 268 268 162 148 21 15 100 352 347 188 162 27 21 21 120 420 400 176 162 29 140 487 467 197 167 37 32 -47-Magnesium as the sulfate appeared to give less constant results than as the chloride. As shown by t y p i o a l experiments, Table 4, some stimulation waa obtained but usually i t was not as great as with the chloride, Manganous ions do not appear to substitute for magnesium ions i n the stimulation of phenylalanine oxidation. Table 5. Slight i n h i b i t i o n of the oxidation was noticed i n some experiments* Effect of pH on the stimulation: Comparison of the stimulation obtained at various pH's 6* 7, 8 , (Pig. 7) showed the greatest % stimulation to be obtained at pH 6, with less at pH 7, and s t i l l less at pH 8. Oxidation at pH 5 was found to be n e g l i g i b l e . Sinoe Gale (i940) has shown the type of amino acid breakdown to be markedly influenced by pH, i t seems probable that the processes operative at the more aoid pH were stimulated by magnesium ions* (On the other hand, i t i s possible that, since the lower pH's were more "unfavourable" for oxidation, magnesium ions made the conditions more "favourable")* Activators: To determine i f magnesium was acting as an activator i n the breakdown of phenylalanine, two d i f f e r e n t organio stimulators were added with and without magnesium ions. Pig. 8. I f greater % magnesium stimulation were obtained i n the presence of the activator, i t would suggest that -48-Table 4 Stimulation of dl-phenylalanine oxidation Time (mine.) MgCl 2.7H 20 or Mg304.7H20 oono. ppm. 20 40 60 80 100 120 140 160 180 200 220 by added Mg304.7H20 Bxpt. 1 C l 2 5 16 33 26 42 56 25 14 S0 4 5 24 26 51 47 0 9# 11* S0 4 50 21 36 36 58 69 17 7 7 % atimulation Bxpt. 2' C^2 5 35 36 39 47 60 42 33 S0 4 0.5 0 3 5 6 7 7 7 30 4 5 0 0 3 11 17 20 11 S0 4  50 16 22 19 26 29 35 25 Bxpt. 3* C l 2 5 36 53 70 70 73 55 41 29 21 30 4 5 4 10 12 8 17 18 21 21 21 # - # i n h i b i t i o n ## - endogenous not subtracted. -49-Table 5 Effect of Mn on oxidation of dl-phenylalanine aa fc stimulation Time -mina. . Expt. 1 WW Expt. 2 ## Expt. 3 HgCl2.7H20 or Mg Mn Mn Mg Mn Mg Mn i!nCl2 oonc. ppm. 5 0.5 5 5. 0.5 5 0.5,5,50,500 20 28 12 12 36 0 7 a l l Inhibited 40 8 0 0 52 0 60 15 0 0 70 3 80 17 12* 0 70 0 100 22 0 7# m 72 0 43 a l l i n h i b i t e d 120 26 4* 9* 66 3 81 tt i t 140 18 4# 14* 40 3 L10 i * « 160 12 Z* 12* 29 7 180 5 1* 13* 21 9 L60 n n 200 1 z* 14* : -# - % i n h i b i t i o n ## - endogenous not s u b t r a c t e d . No added Mg 5 ppm. MgClg .7 f i20 pH 6-pH 7 -pH 8 -Figure 7: E f f e c t of p a on magnesium stimulation of phenylalanine oxidation. 150 - F i l -Ul. 0 z y g e n 120 100 mins • 60 12 0 - phenylalanine plus Mg It Fl Ft It 160 200 plua thiamin - 2 mcgma./cup rt n rt • pyridoxamine-pyridoxine (4 mcgma./cup ea.) rt rt ft Figure 8: Effeot of added thiamin and pyridoxamine-pyridoxine on stimulation of phenylalanine oxidation by added magnesium ions. -52-magneaium waa s p e c i f i c a l l y a c t i v a t i n g the coenzyme i n the breakdown of the amino aoid. I t ia aeen that, although both thiamin (2 micrograma per oupf and a mixture of pyridoxine and pyridoxamine (4 micrograma per oup eaoh) stimulate the oxidation, the percent magnesium ac t i v a t i o n ia no greater with the aotivatora than without them* Inhibitor a : Araenite and semioarbazide are oommonly used as keto f i x a t i v e a . I f the oxidation of phenylalanine passed through a keto compound and i f the magneaium atimulation were involved i n reactions occurring before the formation of the ketoocompound, the "magnesium effedt" should not be decreased i n the presence of the i n h i b i t o r . Tables 6, 7 and 8* show the action of arsenite, aemioarbazide and oyanide on the oxidation of phenylalanine i n the presence and i n the absence of added magnesium ions. The magnesium stimulation ia aeen to be greater i n the preaenoe of aemioarbazide. Araenite, on the other hand, i n h i b i t e d the oxidation completely i n both oases. This suggesta that arsenite may combine with magneaium iona as found by Hoohe et a l (1942) with arsenate* Cyanide did not appear to i n h i b i t the oxidation. Table 9* -53 Table 6 Magneaium effeot on phenylalanine oxidation i n the presence and abaenoe of araenite (1.8 " u moTea) Time mina. No Araenite No Mg Mg Araenite No Mg Mg 80 5 1 40 17 4 80 37 45 2 0 100 54 71 2 0 120 72 104 7 0 140 94 140 3 0 Table 7 Magneaium effeot on phenylalanine oxidation i n the presenoe and absence of semioarbazide fJUB U mffla.) Time mina • No aemioarbazide No Mg Mg Semioarbazide No Mg Mg 20 27 21 8 40 40 63 59 84 68 60 97 104 37 99 80 142 168 47 114 100 192 224 58 138 120 247 297 68 163 -54-Table 8 Magneaium effect on phenylalanine oxidation i n the presence and abaence of cyanide. Endogenous not su b t r a c t e d not°fnn?bftea:anide o o n i t ; r o l a Time fmins.) No cyanide No Mg Mg Cyanide 1 x 10~3M No- Mg Mg Cyanide 1 x 10"2M No Mg Mg 40 53 120 102 93 99 127 60 111 . 193 167 149 172 210 80 149 260 224 213 230 276 100 190 340 280 280 284 332 120 208 380 318 320 320 380 140 240 415 360 352 364 440 180 : 320 475 460 415 465 530 -55-COHCLUSIOHS The influence of various factors on the magnesium ac t i v a t i o n of phenylalanine oxidation by r e s t i n g c e l l s of Ps. aeruginosa has been determined. Increasing the o e l l concentration was found to decrease the stimulation by added magnesium ions. Cells which were grown i n the presenoe of 0,1% MgClg^HgO were not stimulated by added magnesium ions. Stimulation was obtained over the range of 6-5000 ppm, ( f i n a l concentration) MgCl2.7H20« The 1 isomer of phenylalanine was also stimulated. Six moles of oxygen per mole of substrate were required for the oxidation of 1 phenylalanine whereas only four moles of oxygen per mole were required f or the oxidation of d l -phenylalanine. Stimulation of the oxidation was obtained using four times the substrate concentration o r d i n a r i l y used. Ho t o x i c i t y was apparent at t h i s concentration. I-tyrosine, and dl-valine were stimulated by added magnesium ions i n a manner s i m i l a r to that of the d l -phenylalanine stimulation, Dl-alanine was only very s l i g h t l y stimulated. L-histldine was not oxidized at any appreciable rate either with or without added magnesium ions. The oxidation of dl-serine and 1-glutamio acid was 56 s l i g h t l y i n h i b i t e d by 6 ppm, added MgClg^HgO. Oxidation of dl-phenylalanine waa optimum at pH 8 and deoreaaed down to pH 6, No s i g n i f i c a n t oxidation was obtained at pH 5. Greatest $ atimulation of the oxidation of phenylalanine by magneaium waa obtained at pH 6, Thiamin and a mixture of pyridoxine and pyridostemine atimulated the oxidation of phenylalanine aomewhat. However, no greater $ stimulation by added magneaium ions waa obtained i n the preaenoe of these a c t i v a t o r s . Araenite (1,8 umols./oup) i n h i b i t e d the oxidation both with and without added magneaium. Semioarbazide gave a 50$ i n h i b i t i o n , the i n h i b i t i o n being a l i g h t l y leas i n the preaenoe of magneaium iona. Cyanide did not i n h i b i t the oxidation at either 1 x 10"2M or 1 x 10"3M ( f i n a l concentration). -57-PARI 3 MECHANISM OF STIMULATION OF PIIENYLALANINE OXIDATION  BY ADDED MAGNESIUM IONS Sinoe dl-phenylalanine and 1-tyrosine oxidation were foundto be stimulated by added magnesium ions, i t was hoped that by a consideration of the enzyme systems known to be stimulated by magnesium together with a consideration of the possible methods of attack of the amino aoid molecule and the possible intermediate compounds formed, further experimentation would be suggested which would explain the stimulation of the oxidation of these amino acids by magnesium. Enzyme systems known to be stimulated by magnesium ions Numerous references have been made i n the l i t e r a t u r e to magnesium stimulation of phosphatases. Jenner and Kay (1931) obtained stimulation of erythrocyte al k a l i n e phosphatases (hydrolyzlng glyoerolphosphate) by magnesium ions. I n t e s t i n a l a l k a l i n e phosphatases described by Roohe (1943) were also stimulated by magnesium. Aoid phosphatase i n erythrocytes (King et a l , 1945) and aoid phosphatase -58 preparations from l i v e r and kidney (Bamann, 1934) were not stimulated by magnesium. Loss of a c t i v i t y of an a l k a l i n e erythrocyte phosphatase by incubation with buffer at 37°C* was found by Nagamma and Narayana (1948) to be overcome by magnesium ions. Pett and Wynne (1933) have observed stimulation of b a c t e r i a l phosphatases i n CI. aoetobutylicmn and Propbact. j e n s e n i i . Alpha and beta glycerol phosphatases and hexose dlphosphtases were markedly accelerated by optimum m a g n e s i u m f e o n o e n t r a t i o n B which was found to be Q, Mg 2.5 where QMg i s the negative log. of the molar concentration of magnesium ions. Various enzymes i n the Bmbden-Meyerhoff system involving phosphorylation have been found to be activated by magnesium. Phosphorylase, phosphoglucomutase, hexokinase and enolase a l l require magnesium ions f o r a c t i v a t i o n (Barron, 194$. The enzyme enolase was c r y s t a l l i z e d by W arburg and C h r i s t i a n (1941) and found to contain magnesium. Lohmann (1935) using a dialyzed crab muscle extract found that magnesium sal t s allowed the s p l i t t i n g of the second phosphate group from adenosine diphosphate to give adenylic adid. A rat l i v e r suspension which oxidized saturated f a t t y acids was shown by Lehninger (1945) to require adenylic acid, inorganic phosphate, cytochrome C and magnesium ions. Deamination has been variously stated to be stimulated -59-and i n h i b i t e d by magneaium iona. Stoner and Green (1946) found magneaium (0.0026M magneaium ion) to be a powerful i n h i b i t o r of the deaminasea of adenosine triphosphate found i n rat muscle. Increasing the concentration of magnesium from one mg. to seven mg. per 100 ml. deoreased the dsemination from 80% to 45%* Blelachowaky (1946) has suggested that the overcoming of the oardio-vascular effects of certain purine derivatives by magnesium might involve a deoreased rate of deamination. On the other hand, Jaoobsohn (1935) et a l reported that magnesium ions displaced the system aspartio ? fumario -t- ammonia to the r i g h t . A non-enzyme reaction, the deamination of glycine with hydroxy-hydro-quinohe or resor c i n o l as catalyst was shown by Kirsch et a l (1933) to be activated by 0.001 - 0.002M calcium, magnesium, strontium or barium chloride. The stimulation of serine deaminase by zinc, manganese or magnesium as reported by Binfcley (1943) has been cit e d previously. Berger and Johnson (1939) and Boohe et a l (1947) have found i n t e s t i n a l peptidase to be activated by magneaium or manganese ions. The formation of glutamic acid from i s o c i t r a t e i n tissues has been reported by Adler et a l (1939) to be oatalyzed by an enzyme requiring magnesium ions f or a c t i v a t i o n . Many decarboxylase systems have been reported to be stimulated by magnesium ions. Aooording to Lohmann and 60 Schuster (1937) magnesium was found to stimulate the decarboxylation of pyruvate by washed yeast.In the presence of diphosphothiamin. The o r y s t a l l i n e enzyme carboxylase was isolated by Green (1940) and was shown to be a complex consisting of diphosphothiamin plus magnesium plus a speoifio protein. Krampitz and Workman (1941) found oxalacetate decarboxylation from M. lysodelkticus to be stimulated by magnesium but i n this case, neither co-carboxylase nor thiamin were required. Magnesium added to -depleted c e l l s i n thiamin and acetate wasjfound by Quastel and Wesley (1942) to increase the oxygen uptake. Magnesium and potassium increased the oxidation of l a c t a t e even when no B i was added. An i n t e r e s t i n g function of magnesium has been reported by Pollock and Wainwright (1948). U i t r i t a s e adaptation i n a growing coliform culture was found to be stimulated by the addition of amino acids and magnesium ions. The adaptation was much more rapid than generalized o e l l growth. Geiger (1947) found the oxidation of amino acids by fumarate-grown oel l s of E. c o l l to be f a c i l i t a t e d by phosphate, magnesium and cocarboxylase. Summing up the known funotions of magnesium i n enzyme systems, magnesium has been shown to be important i n enzyme systems involving decarboxylation, deamination, phosphorylation, breakdown of peptides and i n the formation of adaptive enzymes. -61 Amino Acida Sinoe many reactions involving phosphorus have been found to be stimulated by magneaium and magneaium has been found to atimulate the oxidation of phenylalanine and tyroaine, the p o a s i b i l i t y of phosphorylation of amino acida waa auggested. Umbreit (1949) has stated that although phos-phorylation of amino acida has not been shown to occur, phosphorylated amino aoids have been found i n tiaauea (Binkely, 1 9 ^ 4 4 ) . Evidence againat phoaphorylation of amino aoids haa been found i n the work of Hotohkiaa (1947). Hi8 experiments with washed oell a of Staph, aureua ahowed a decrease i n inorganic phoaphate e a t e r i f i e d when amino acida were added to a glucoae oxidation even though the amino acida were apparently broken down. Ever aince the discovery of the olasaic methoda of breakdown of amino acida, deamination and decarboxylation and the variations depending on the r e l a t i v e amount of oxygen and the organiama concerned, described by Gale (1940), various other pathways ofjamino aoid breakdown have been proposed. Braunatein (1939) haa shown "tranaaminatlon n - the passing of ammonia from an amino aoid to a oompound auoh as oxalacetate to be an important proceaa i n nitrogen metabolism. L i c h s t e i n and Cohen (1945) found strong transamination syatema i n B. c o l l , proteua organiama, •62-Azotobacter, Staphylococci, C l o s t r i d i a , Streptococci and Pneumoooooi. Gedrangolo (1943) haa questioned t h i s system on the basis of lack of a c t i v a t i o n of 1-alanine oxidation by added alpha keto glutarate i n kidney and l i v e r s l i c e s . Blooh (1946) haa ahown acetylation of amino acids i n v i t r o using deuterlo acetic acid; a c e t y l a t i o n waa said to be a normal part of amino acid metabolism. The decomposition of one mole of dl-phenylalanine by resting c e l l s of Pa. aeruginosa grown at pH 7*8 waafound by Webster and Bernheim (1936) to require f i v e molea of oxygen and to give off two molea of CQ2. The oxidation of the 1 iaomer required 6^ molea oxygen and gave off 4 molea C02* N o evidence for deamination of the d iaomer waa aaid to be found but the t h e o r e t i c a l amount of ammonia wa8 recovered from the deamination of the 1 iaomer at the end of 4 hours. The d iaomer alone waa not deaminated. However, the ammonia produced by deamination of the d l aoid was 1.5 times that which would be expected based on the complete deamination of the 1 isomer only. The oxidation of phenylalanine by "resting'* c e l l a of Proteus vulgaris waa found to require one mole of oxygen per mole of the amino aoid according to Bernheim et a l (1936). Ihe natural iaomer only waa attacked. Two possible methoda of i n i t i a l attaok of the amino aoid phenylalanine have been found by varioua workers. The keto aoid formed by the oxidative deamination of phenylalanine has -63-been i d e n t i f i e d both i n vivo and i n v i t r o , i n animal tissues and i n b a c t e r i a l cultures. The primary oxidation of the r i n g with the formation of tyrosine has been considered the i n i t i a l reaction by some workers. 3tudiea on the accumulation of phenylpyruvic aoid i n the mammalian catabolism of phenylalanine were made by Chandler and Lewis (1932) and Sealock et a l (1941). Chandler and Lewis (1932) considered that the accumulation of phenylpyruvic acid i n the urine of rabbits fed phenylalanine indicated that the benzene r i n g i s broken less r e a d i l y than generally assumed. Sealock et a l (1941) found ascorbic acid to be involved i n the metabolism of phenylalanine. The accumulation of phenylpyruvic aoid i n vitamin C-defioient guinea pigs was'found to be prevented by feeding of vitamin C. The accumulation of the keto acids from tyrosine and phenylalanine i n premature infants was prevented by ascorbic aoid according to Levine et a l (1941). Henriokson and Closs (1938) reported large amounts of phenylpyruvic acid from phenylalanine i n cultures of B. proteus and S a l . morganii. Uemura (1942,1944) showed the formation of the keto acid, then the hydroxy acid according to the scheme of Ueubauer (1911) from d l phenylalanine decomposition by microorganismsj Simmons et a l (1947) were able to develop mutant strai n s of E. c o l l which required phenylalanine or tyrosine. In t h i s case, phenylpyruvic substituted f o r -64-phenylalanine but not for tyrosine and tyrosine was therefore not considered an intermediate i n phenylalanine breakdown. On the other hand, data have been accumulated showing the oxidation of phenylalanine to tyrosine to be the primary step i n the breakdown. Smbden and Baldes (1913) did not considerthe formation of phenylpyruvic acid to be the primary reaction i n catabolism of phenylalanine by dog l i v e r . (Tyrosine was isolated i n the perfusion f l u i d when phenylalanine was perfused through the tissue. Twenty to t h i r t y percent of the tyrosine of the tissues of the i n t e r n a l organs i n rats was found to be formed from d l phenylalanine using the compound containing heavy hydrogen. Even with excess tyrosine i n the d i e t , 13% of the tyrosine of the tissues was found to have arisen from phenylalanine. An hydroxy phenyl compound formed from 1 phenylalanine inoubated with l i v e r s l i c e s was concluded to be tyrosine by Bernheim and Bernheim (1944). Using the recent technique of comparative analogue-me t a b o l i t e growth i n h i b i t o r s , i n t h i s oase B-2-thienylalanine, Beerstecher and 3hive (1947) concluded that i n E. c o l l . keto acids were not intermediates i n phenylalanine oxidation since phenyl pyruvate was i n e f f e c t i v e i n preventing the}toxicity of B-2-thienylalanine. The oxidation of phenylalanine to tyrosine whs stated to be i r r e v e r s i b l e with E. c o l l . The primary decarboxylation of ah ;amino acid i n our -65-experlmenta i a u n l i k e l y since Gale (1943) has shown the pH range of a c t i v i t y to be pH 4 to pH 6 with no a c t i v i t y at pH 7. Taylor and Gale (1945) found decarboxylases from bacteria f or l y s i n e , tyrosine, h i s t i d i n e , arginine and ornithine. The optimum pH was 4.25-5.25* MoGilvery and Cohen (1943) obtained aoetone powder preparations from 3o. faeoalia which deoarboxylated t y r o i s i n e and phenylalanine. The rate of decarboxylation of phenylalanine was l/lOO the rate of tyrosine decarboxylation. Also, Anderson (1947) found decarboxylation to follow deamination i n most cases with the genua Proteus. Gurin and Delluva (1947) showed adrenalin synthesis from phenylalanine by decarboxylation i n the animal body using labelled phenylalanine. Gale (1942) has studied the b a c t e r i a l decomposition of amines, the compounds which would be formed when amino aoids are deoarboxylated. The optimum pH for amine oxidation was found to be a l k a l i n e . With Ps. pyooyanea. the enzymea for the oxidation of tyramine and hiatamine were adaptive. The oxidation of benzylamaine waa very slow. Although, from the oxygen uptake, i t can be aaaumed that the benzene ri n g i s broken i n the oxidation of phenylalanine, the mechanism of the s p l i t i s a t i l l obscure. Bernheim and Bernheim (1944) concluded that, beoause of the disappearance of estimable OH groups, the r i n g of tyramine was s p l i t by muscle enzymes of the r a t . Oxidation of the benzene r i n g by -66-a o i l bacteria waa studied by Evans (1947). Cateohol and hydroxy benzoic aoid were found aa intermediate products, then orthobenzoguinone, dihydroxy benzoic acid, a keto acid a and formic acid. He ooncluded that tyroaine waa deaminated before a p l i t t i n g of the r i n g . Traetta-Moaoa (1910) found pOH phenyl propionic, benzoic acid and benzene to be produoed by B. pyocvaneus i n an inorganic medium containing tyrosine. According to Stone et a l (1949), the fermentation of tyrosine by marine baoteria involves oxidation of the side ohain with the progressive removal of carbon-dioxide. The compounds formed were pOH phenyl pyruvic a c i d , pOH phenyl acetic aoid, p c r e s o l , pOH benzaldehyde, pOH benzoic aoid and phenol. When tyramine was used, only traces of a phenolio substance could be found and no p cresol waa detected. I n i t i a l decarboxylation of tyrosine waa therefore conaidered u n l i k e l y . Sinoe phenol apparently could be formed only from tyroaine and not from intermediatea, a breaking of the r i n g from the side chain aa i n indole formation from tryptophane, waa postulated. The breakdown of phenylglycine^; a homologue of phenyl-alanine , by yeast was investigated by Neubauer and Fromherz (1911). Phenylglyoxylic a c i d , mandelic aoid and benzyl alcohol were deteoted i n a growing culture. -67-METH0D3 Bac t e r i o l o g i c a l ; The Warburg technique as described i n the previous section was used* Carbon-dioxide was determined by the " a c i d - t i p " method desoribed i n Umbreit et a l (1945). In the determination of phosphorus uptake, 0.5 cc* of M/30 UaHCOg and 0*6 oo. of ZH2po4 (equivalent to 130 micros grama of phosphorus per cup) were substituted i n thejcups for the Sorenson's phosphate buffer. Carbon-dioxide --a i r (about 10% COg) aymosphere was used to bring the pH to 7.2. Cupa were removed at 0,10,20,40,60,80*100 and 120 minutes and 0.06cc. 100% t r i c h l o r a c e t i c acid were added to stop the reaction, p r e c i p i t a t e the o e l l s and extract the soluble phosphorus from the c e l l s . The o e l l s were removed by oentrifugation and phosphorus was determined on the supernatants. For the determination of ammonia, 3N HC1 was tipped i n from the f l a s k sidearm and the contents of the cups were removed with a Pasteur pipette for analysis* For the determination of intermediate compounds, large volumes of supernatant were required. One hundred cc. quantities of substrate, resting c e l l s , phosphate buffer, i n the proportions as used i n the Warburg flasks were used i n chemically cleaned one l i t r e f l a s k s (Erlynmeyer). The flasks were incubated for 2 hours at 30°C. on a shaking -68 machine. The c e l l a were centrifuged o f f and the aupernatanta were vacuum d i a t i l l e d down to a concentration approximately 100 times that of the i n i t i a l concentration. The oompounda preaent represented those produoed from 30 mg. of the amino ac i d . Chemical: Ammonia was determined by a modification of the micro-diffusion technique of Conway and Byrne (1933). Ordinary p e t r i dishes were uaed with watoh glasses mounted on p l a s t i o i n e as the inner vessels. The test solutions, containing not more than 100 micrograms of ammonia, were added to the outside seotion. One cc. of O.ltf H 2 S O 4 was pipetted onto the watchglass. The edgea of the dish were greased to assure an a i r - t i g h t f i t between the two halves. Two cc. of saturated K 2C0 3 were added to the outside section and the top of the p e t r i was quickly placed on the bottom. The dishes were incubated at 37°0. for two hours a f t e r which time the l i q u i d was removed from the watch glass with a fine bore pipette and the watch glass was rinsed several times with d i s t i l l e d water. Thesaolutions were Neaalerized i n either 25 or 50 0 0 . volumetric f l a s k s . Standard solutions of ammonium sulfate were run as oontrols. The phosphorus determination uaed was that of King (1932) baaed on the color produced by the i n t e r a c t i o n of molybdate, aminonaphthosulfonic aoid and phosphorus. Standard curves were run along with the test solutions. ^69 Phenylalanine waa determined by the method of Blook and B o i l i n g (1940) aa described i n Part 1. Chroma to graphy; 5)he c a p i l l a r y aaoent method aa described by Williama and Kirby (1948) waa uaed. A butanol - acetio acid mixture (405 oo* butanol,10 cc, acetio aoid, 450co. water) was the solvent used. Following oompletion of i r r i g a t i o n , the solvent was evaporated o f f by heat and the sheets were then sprayed with nlnhydrin s o l u t i o n (ninhydrin 0.1$ i n butanol). Unknowns and knowna (oompounda which would possibly be present i n the breakdown of phenylalanine and which would give a ninhydrin reaction - tyrosine, phenylethyl amine, tyramine) were run i n p a r a l l e l . -70-EXPERIMENTAL AND DISCUSSION In an attempt to f i n d a aatiafaotory explanation of the stimulating a c t i o n of magneaium iona on the oxidation of phenylalanine, i t waa considered highly desirable to investigate further the influence of t h i a ion on possible enzymatic changes which the amino aoid or i t a breakdown products might undergo. Since magnesium has been found to be conneoted with so many enzyme systems involving phosphorylation, i t was deolded to determine i f phosphorus was involved i n phenylalanine breakdown. Over a two hour incubation period i n the Warburg, no phosphate uptake was detected. Table 1. The fact that oxidation and magnesium stimulation ooourred i n the absence of added phosphate, that i s , i n Ringer's solution and i n saline ( F i . g 1) suggested also that phosphate was not involved i n the breakdown. The part played by magnesium i n the formation of the adaptive.enzyme n i t r i t a s e (Pollock and Wainwright, 1948) suggested a s i m i l a r role of magnesium i n the formation of the adaptive enzymes oxid i z i n g phenylalanine. The oxidation of glucose by c e l l s grown on acetate i s known to be an adaptive process (Ney, 1948). I f magnesium stimulated the formation of the adaptive enzymes, the rate of glucose breakdown by acetate-grown c e l l s should be greatly lnoreased 71 Table 1 Uptake of phosphorus during phenylalanine oxidation • Time. mina • Phosphorus micro gra oma /cup 0 106 1G 112 20 132 40 125 60 126 80 119 100 139 120 132 Gup oontenta; M/30 NaHC03 0.5 oo. ( f f f^mi cr ograma 0 ' ^  phosphorus/oup) phenylalanine 0.2 " (sldearm) MgCl2.7Hg0 (5 ppnu f i n a l oonoi) 0*3 " Cella - 40 x saline suapens. 0.5 " Atmoa,: approx. 10#C02 i n a i r to give pH 7.0 No KOH i n centre w e l l Figure 1: E f f e c t of magneaium on oxidation of dl-phenylalanine i n absence of phosphate. -73-by magnesium, This i s shown to be the case i n Fig. 2. An attempt was made to remove the magnesium stimulation of phenylalanine oxidation by preadaptation of the culture to phenylalanine. Two methods were t r i e d . The culture was transferred d a i l y seven times i n the growth medium containing 0.2% phenylalanine before f i n a l transfer into the medium used f o r the growth of r e s t i n g c e l l s which contained 0.1% phenylalanine i n addition to the usual constituents. C e l l s were also grown i n the same medium (containing phenylalanine), the transfer being taken from the usual g l y c e r o l peptone medium. Phenylalanine oxidation with these c e l l s was compared with that obtained with the usual o e l l s (Fl.g 3). The stimulation with both sets of phenylalanine-grown c e l l s was as great as obtained with those grown i n the usual medium. No adaptation was apparent using either of the c e l l suspensions, The stimulation of phenylalanine oxidation by magnesium ions doesNaot-appear to be oaused by a greater rate of adaptive enzyme formation. The disappearance of phenylalanine was found to be greatly aocelerated i n the presence of magnesium. At the end of four hours, the amount of substrate used with magnesium added waaalmost double that used i n the absence of k] magnesium (Table 2). I t i s in t e r e s t i n g to notice a tendency of the r a t i o (amino aoid used /oxygen uptake) to deorease during the course of the reaction." This suggests that the Figure 2: Effect of added magneaion iona on adaptation of acetate grown c e l l a to glucose oxidation. Figure 3: Effect of added magneaium iona on oxidation of phenylalanine hy phenylalnine-grown c e l l a . 76-Table 8 Effeot of added magneaium Iona on oxidation and diaappearanpe of phenylalanine ( 1 . 2 mg. phenyls1 ./cup) Time. mina. No added magnesium 6 ppm. added MgCl 2 .7Hgl u l . 0 2 P.A. uaed mg. P.A. "W u l . 02 P . A . uaed mg. P . A . 02 60 76 0 . 4 5 5 . 9 74 -120 175 0 . 3 5 2 . 0 225 0 . 5 5 2 . 4 180 260 0 . 5 6 2 . 1 410 0 . 8 1 . 9 210 L 303 0 . 5 5 1 . 8 498 l . o 2 . 0 Effect of added magneaium on deamination  of phenylalanine* 1*2 mg* phenylal. /cup as micrograms ammonia/ oup. Time houra• Endogenous Phenylal* Phenylal* with 6 ppm* Mg01g.7H20 2 30 45 40 3 50 75 55 4 40 67.5 65 -78-ratea of i n i t i a l attack of the moleoule and I n i t i a l atages i n the breakdown are greater than thoae further along i n the ohaln of reaetiona. Very l i t t l e ammonia, r e l a t i v e to the possible t h e o r e t i c a l t o t a l waa found (Table 3).. At the end of four houra, only about 25$ of the t h e o r e t i c a l waa produced. Aa demonstrated by Stoner and Green (1946), magneaium iona were found to deoreaae a i i g h t l y the rate of deamination of the amino aoid. Thia being the case, i f the breakdown goea according to thia method, i t ia apparent that the atimulation of the breakdown of the intermediate, whatever i t may be, ia greater than i a shown by the o v e r a l l atimulation of oxygen uptake of the amino acid. In view of the greatly inoreaaed atimulation of phenylalanine oxidation at pH 6 aa compared with that obtained at pH 8, a difference i n the type of reaction occurring at theae d i f f e r e n t pH's waa aought. The E.Q.*a (ratio of carbon-dioxide evolution to oxygen uptake) were oompared at pH 6 and pH 8. Table 4. The H.Q,. at pH 8 waa i n a l l caaea about double that obtained at pH 6 ( i n d i c a t i n g r e l a t i v e l y greater CGg production at pH 8). In the presence of magneaium however, the E.Q,. at pH 6 and pH 8 were the same approximating thoae obtained with pH 8 i n the absence of added magnesium. A fundamental difference i n the type of breakdown ooourring was indicated. 3tt pH 6, lesa deamination and greater 79 Table 4 Effect of magneaium lona on decarboxylation of -phenylalanine (5 ppm. MgClg.7HgO) u l . Og pH 6 pH 8 Expt. 1 2 3 1 2 3 u l . COg 48 54 846 128 > 225 386 U l . Og 85 86 258 166 185 185 B.Q. C 1.56 0.62 0.96 0.77 1.28 1.76 Mg added pH 6 pH 8 u l . COg 134 137 u l . Og 133 144 E.Q. 1.00 0.95 pH 7 No Mg Mg added Expt. 1 2 3 1 8 3 u l . COg 99 19 6 91 58 80 u l . Og 97 50 58 99 59 49 0.98 0. 38 0.12 1.02 1.00 0.40 -80-deoarboxylatlon are known to ooour. At pH 8, the opposite i s true. These reactions oannot be a f f e c t i n g the R.Q.'s greatly sinoe they would tend to r a i s e the R.Q. at pH 6 and lower i t at pH 8. The difference i n E.Q. at these two pH's i s therefore apparently unrelated to amino aoid deamination or decarboxylation. Other reaotions tending to lower the R.Q. are those oxidative reactions involving no C0 2 evolution such as r oxidation of the benzene r i n g with the formation of hydroxy-• benzene derivatives or oxidation of the side ohain without the removal of carbon atoms. Reactions which would possibly raise the R.Q,. are those involving COg evolution with or without corresponding oxygen uptake and include s p l i t t i n g the benzene r i n g with subsequent step-wise removal of C0 g or s p l i t t i n g the side ohain. I t i s therefore concluded either that at pH 6, formation of hydroxy derivatives or oxidation of the side chain i s favoured or that at pH 8, s p l i t t i n g of the benzene r i n g or breaking of the side chain i s ooourring at a greater rate. Sinoe the rate of oxygen uptake at pH 8 was usually greater than', at pH 6, the l a t t e r i s probably the case. An attempt was made to d i f f e r e n t i a t e between the type of oxidation occurring i n the presence and absence of magnesium by determination of the R.Q. Table 4. R.Q.'a determined when 100 u l oxygen had been taken up i n both cases, showed no difference. At the end of 50 u l . oxygen uptake, -81-however, not only waa the R.Q.considerably lower than at 100 u l . oxygen uptake, but the R.Q. with no added magnesium was found to be about l/3 that with added magnesium. Magnesium thus appears to be stimulating reactions involving the breaking of the carbon ohain. Anaerobic decarboxylase a c t i v i t y at n e u t r a l i t y was found to be n e g l i g i b l e . Table 5. The decarboxylation of phenylalanine i s therefore concluded to be oxidative. A technique often used i n the determination of Intermediate compounds i s to compare rate of oxidation of possible intermediate compounds with that of the parent substrate. Several oritioisma of t h i s technique have been made. It has been suggested that permeability may be a l i m i t i n g factor. In other words, i f the enzymes breaking down the parent substrate are i n t r a c e l l u l a r , the intermediate compounds may be released and be further broken down within the c e l l . Therefore, an a b i l i t y to penetrate the o e l l membrane i s not a necessary prerequisite for an intermediate compound. Sinoe lack of permeability of the compound may cause lack of oxidation, i t i a apparent that an intermediate may or may not be attacked by the organism. Also, many c compounda are phosphorylated i n the f i r s t stage of attaok with subaequent oxidation of the f i r s t intermediate. I f the organlam i a unable to phosphorylate the intermediate as such, no oxidation of that intermediate w i l l ocour. -82-Table 5 Anaerobic decarboxylation of  of phenylalanine carbon-dioxide oxygen Phenylalanine 1.8 uMoles/oup Phenylalanine 7.2 umOlea/cup Time 180 mins. 90 mine. Endog. -15 -10 Phenylalanine * 7 •" #:-9i-4 Phenylalanine plus Mg. HO £25.0 Gup content a : M/30 Sorensonfs phosphate buffer phenylalanine (sidearm) MgClg .7Ho0 ( f i n a l oono. 5 ppm.) o e l l s - 40x - saline suspension 3N HC1 (sidearm) no KOH 1.5 co. 0.2 " 0.3 * 0.5 " Atmos: nitrogen -83-Thus the f a l l a c y of aaauming lack of oxidation of a possible intermediate compound to indioate that the compound i a not an intermediate, ia apparent. On the other hand, i f a possible intermediate i s attacked, the p o s s i b i l i t y of i t a being i n the breakdown ayatem i a atrongly indicated. But t h i a i n i t a e l f i a no proof and merely conatltutea aupporting data. However, i f the intermediate i a attacked with greater f i n a l oxygen uptake on a molar baais, then i a obtained with the parent aubatrate, i t i a obvioualy not i n the cycle. It aeema reasonable that the f i n a l oxygen uptake of an intermediate oompound one oxidative atep removed from the i n i t i a l compound ahould require one-half mole of oxygen per mole of aubatrate leaa than the f i n a l oxygen uptake of the i n i t i a l compound. Webster and Bernheim (1936) found 5& and 6£ molea of oxygen taken up by the oxidation of one mole of 1-tyrosine and 1-phenylalanine respectively with Pa.  pyocyaneus. We have found (Part 2) that under our conditions, four molea of oxygen and 6 molea of oxygen were required f o r the oxidation of l-tyrosine and 1-phenylalanine respectively. From these two sets of data, i t appears either that tyrosine i a not an intermediate i n phenylalanine oxidation or that an alternative method of breakdown of phenylalanine by Pa.  aerugino8a i s possible. The oxidation of other possible intermediate compounds was determined (Table 6). I t i a seen that neither tyramine^ -84 Table 6 Oxidation of possible intermediate compounds u l . 0 Time mina. Tyramine Bndog. No Mg Mg Phenyletfaylamine ESndog. No Mg Mg Phenylpyruvate Bndog. No Mg Mg 20 40 46 47 40 57 61 66 66 61 48 56 46 49 60 72 77 84 87 84 71 84 72 79 80 88 94 L05 109 105 94 117 100 110 100 100 106 122 128 124 112 150 132 144 120 113 122 144 150 147 133 180 161 173 140 120 130 158 168 168 149 208 188 203 160 186 186 166 230 214 228 -85-phenylethylamine nor phenylpyruvate were attacked at any appreciable rate, either i n the preaenoe or i n the absence of magnesium. I t is concluded either that these compounds are not intermediates i n the breakdown of phenylalanine by Ps. aeruginosa or that they are not able to penetrate the c e l l membrane. It was thought that magnesium might i n some way be allowing oxidation of the benzene r i n g . A series of representative compounds containing a phenyl group were tested i n the Warburg f o r oxidation with and without added magnesium. Magnesium had no effeot on the oxidation of any of the compounds t r i e d except phenyl glycine where a tendency to t o x i c i t y apparently waa overcome i n the presence of magnesium. Hydrocinnamic acid was the only compound t r i e d where the oxygen uptake exceeded that of the endogenous and even here, the oxidation waa r e l a t i v e l y s l i g h t . Table 7 Neither tyramine, phenylsthylamine nor tyrosine could be detected as a product of oxidative breakdown using the chromatographic technique w i t h the ninhydrin reaction. Since t h i s reaction i s sensitive to about O.lmg./cc, i t appears that these compounds are either present i n lesser quantities (1/300 of substrate) or are not intermediates i n the breakdown of phenylalanine. It i s considered very l i k e l y that more than one method of breakdown of the phenylalanine molducle by Ps. aeruginosa i s occurring. I f this i s true, the d i f f i c u l t y of detecting an intermediate compound i s consequently increased. -86-Table 7 Effect of added magneaium iona on the oxidation  of^phenyl compounda» Time mina. Endog. Phenyl aoetate No Mg Mg Phenyl glycine No Mg Mg hydrocinnamio acid No Mg Mg mandelic aoid No Mg Mg 20 52 54 48 42 52 70 48 55 51 40 91 91 86 70 95 120 82 100 90 60 132 126 123 104 135 150 118 140 125 80 165 154 157 133 168 220 146 173 156 100 196 188 186 156 199 260 173 208 181 120 222 212 210 180 226 294 195 238 206 •87 CONCLUSIONS The effect of added magnesium ions on some mechanisms of phenylalanine breakdown by Ps. aeruginosa has been determined. According to the data obtained, phosphorylation i s not H essential to either phenylalanine breakdown or magnesium s stimulation of phenylalanine breakdown by Pa. aeruginosa. Disappearance of substrate was greatly increased i n the presence of magnesium ions. The r a t i o amino acid used to oxygen taken up increased with time i n d i c a t i n g a la g i n the rate of breakdown of some of the l a t e r Intermediate compounds formed. Deamination of phenylalanine was s l i g h t l y i n h i b i t e d i n the presence of magnesium ions. The R.Q.. (r a t i o of COg evolved to oxygen taken up) at pH 8 was greater than that obtained at pH 6, possibly i n d i c a t i n g that the carbon chain i s s p l i t more r e a d i l y at pH 8. The same type of R.O.. was obtained i n the presence of magnesium at both pH's. At n e u t r a l i t y , added magnesium increased the R.Q,. I t is concluded that both a higher pH and added magnesium stimulates breaking of the carbon ohain. No anaerobic decarboxylation of phenylalanine ocourred. No intermediates could be detected either by chromatography -88-or by oxidation i n the Warburg. 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