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Temperature acclimation effects on proteins in the eurythermal fish Gillichthyes mirabilis Kuo, Freiya 1974

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TEMPERATURE ACCLIMATION EFFECTS ON PROTEINS IN THE EURYTHERMAL FISH GILLICHTHYES MIRABILIS B.A. University by FREIYA KUO of C a l i f o r n i a , Berkeley, 1967 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE.DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT of ZOOLOGY We accept this thesis as conforming to the required standard: THE UNIVERSITY OF BRITISH COLUMBIA September 1974 In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permission. The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Depa rtment ABSTRACT The effect of temperature on biochemical adaptation i n the eurythermal f i s h G i l l i c h y t h e s m i r a b i l i s , long jaw mudsucker, was studied with respect to two questions: (a) How much restructuring of ov e r a l l tissue proteins occurs with acclimation to cold and warm temperatures, and (b) how does the nature of protein restructuring relate to eurythermy? Much l i t e r a t u r e exists showing temperature acclimation induces changes i n proteins and enzyme functions, and that these changes may be short term or long term induction effects. However, no statement has been made for the extent of ov e r a l l protein pattern restructuring that may occur with thermal acclimation. In the eurythermic species studied, no evidence arises for major changes i n protein composition with thermal acclimation. The results are considered i n a discussion (a) of some of the environmental l i m i t s imposed upon this species, (b) of evolution under eurythermic and stenothermic conditions i n selective u t i l i z a t i o n of gene products for adaptation on the molecular l e v e l , and (c) of some characteristics of enzymes important to their functional differences, as found by other investigators. i i i TABLE OF CONTENTS Page Abstract i i L i s t of Figures y L i s t of Graphs yi Acknowledgements v i i I. INTRODUCTION 1 I I . MATERIALS 4 1. L i s t and sources of chemicals used 4 2. Experimental materials 5 I I I . METHODS 1. Acclimation 6 2. Polyacrylamide gel electrophoresis 7 a. r e c r y s t a l l i z a t i o n of monomers 7 b. stock solutions 8 c. working solutions and preparation of gels 8 3. Sodium dodecyl sulfate gels 11 4. Staining and recording of protein bands i n SDS and polyacrylamide gels 12 a. TCA f i x a t i o n of proteins 12 b. staining of proteins 12 c. destaining and storage 12 d. photography and densitometry graphs of gels 13 5. Starch gel electrophoresis 13 a. procedures for a discontinuous buffer system 14 b. s p e c i f i c enzymes studied 15 iv 6. Tissue preparation and c e l l u l a r fractionation 17 a. tissue preparation 17 b. subcellular fractionation 18 c. polyribosome i s o l a t i o n 20' IV. RESULTS 21 1. Acclimation 21 2. Gel electrophoresis 23 3. Polyribosome and monoribosome p r o f i l e 41 V. DISCUSSION 42 1. Electrophoretic results and tissue comparisons 42 2. Acclimation adaptations 44 3. Protein and enzymic functional adaptations 45 APPENDIX A 49 1. Polyacrylamide gel electrophoresis 49 2. Sodium dodecyl sulfate acrylamide gel electrophoresis 52 APPENDIX B: Densitometry graphs of acrylamide gels 54 BIBLIOGRAPHY 67 LIST OF FIGURES Figure Page 1 Surface sea water temperatures 22 2 Gillichythes m i r a b i l i s muscle proteins 27 3 Gillichythes m i r a b i l i s brain proteins 28 4 . Liver proteins short term freez storage 29 5 Liver proteins long term freez storage 30 6 Starved 28°C stressed Gillichythes m. tissues 31 7a 13 days acclimation 32 7b 20 days acclimation 32 8a 31 days acclimation 33 8b 38 days acclimation 33 9 45 days acclimation 34 10 52 days acclimation 35 11a Low speed v.s. high speed post mitochondrial supernatant 36 l i b Mitochondria triton-x-100 soluble proteins 36 12 Acid gels of l i v e r tissue subcellular fractions 37 13 Sodium dodecyle sulfate acrylamide gels of mitochondria 38 14 Fresh 24°C trout tissues; l i v e r , muscle, brain 39 15 Frozen trout tissues; l i v e r , muscle 40 LIST OF GRAPHS Graph Densitometry graphs of polyacrylamide gels: Page I Gillichythes m i r a b i l i s muscle 55 II Liver, non-frozen tissue 56 III Liver, one week frozen stored tissue 57 IV 28°C starved stressed - l i v e r 58 V 28°C starved stressed - muscle 59 VI 28°C starved stressed-brain 60 VII 13 days acclimation 61 VIII 20 days acclimation 62 IX 31 days acclimation 63 X . 38 days acclimation 64 XI 45 days acclimation 65 XII SDS gels of mitochondria 66 ACKNOWLEDGEMENTS I would l i k e to thank Dr. Peter W. Hochachka for being my supervisor, for his support through some very d i f f i c u l t times, and for his wonderful s p i r i t and friendship, which have a l l made this work possible. I would also l i k e to thank Dr. George N. Somero for his generous help, and for the opportunity to work i n his laboratory at the Scripps Institute of Oceanography, University of C a l i f o r n i a , San Diego; I am greatly indebted to him for these and more. To my parents, who helped spark the love for nature and an interest for the pursuit of knowledge, I thank them for a l l t h e i r love and in s p i r a t i o n . F i n a l l y , my thanks to the Fisheries Research Board of Canada and the Zoology Department of the University of B r i t i s h Columbia who have helped support me i n this study. 1 I. INTRODUCTION The study of biochemical adaptation to environmental temperature changes i n poikilotherms has indicated various mechanisms for maintaining metabolic homeostasis. Discussions of e a r l i e r l i t e r a t u r e have been given by Bulluck (1955), Fry and Hochachka (1970), and more recently by Hochachka and Somero (1971, 1973), and Hazel and Prosser (1974). The a b i l i t y to maintain stable metabolic rates over wide ranges of temperature changes, diurnally and seasonally, has been attributed to a number of "strategies" whereby temperature operates as a modulator, affecting enzymic a c t i v i t y either d i r e c t l y or i n d i r e c t l y , and with immediate compensatory or longer seasonal acclimation effects. Direct modulation of enzyme a c t i v i t i e s during acclimation has been indicated by such phenomena as (a) functional interconversions of one enzymic form (Somero, 1969; Behrisch § Johnson, 1974a), (b) induced changes i n isoenzymes variants (Baldwin £ Hochachka, 1970; Hochachka § Lewis, 1970; Hochachka § Clayton-Hochachka, 1973), and (c) induction of s p e c i f i c regulatory enzymes resulting i n major restructuring of metabolic pathways (Hochachka, 1968). Temperature may also impose numerous effects i n d i r e c t l y on enzyme a c t i v i t y by affecting c e l l u l a r i o n i c environments and enzyme-ligand a f f i n i t i e s (Behrisch § Johnson, 1974b; Moon, 1972). A l l these influences would necessarily have complex effects upon metabolic integrations and c e l l u l a r energetics. Studies of protein synthesis have indicated acclimation-induced changes i n rates of protein synthesis (Das § Prosser, 1967; Haschemeyer, 1969a § b). These, and results from isoenzyme studies provided ground 2 for the proposal that economy of c e l l u l a r energetics would favor production of new variants of an enzyme possessing apparent Km (enzyme a c t i v i t y at substrate concentrations giving one h a l f the maximal reaction velocity) properties more e f f i c i e n t at new a c c l i -mation temperatures rather than producing larger quantities of the same enzyme. A consideration of these facts led to the question: To what extent does restructuring of proteins occur i n acclimation? Since a systematic investigation of a l l regulatory enzymes would have been a ponderous project, the question was approached by subcellular fractionation and studies of overall protein pattern d i s t r i b u t i o n s using acrylamide and sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis. Studies were also performed on polysomes and monosomes to determine i f any correlations existed between protein synthesis and acclimation temperatures. The eurythermal f i s h Gillichthyes m i r a b i l i s (long jaw mudsucker) was selected for the studies because of i t s great capacity for metabolic adaptation, which i s suggested by the tolerance to wide temperature ranges and other physical factors encountered i n i t s normal estuarine habitat (Todd § Ebeling, 1966; Weisel, 1947). In attempting to answer the question put forth for t h i s research, the results are discussed with respect to the eurythermic nature of Gillichthyes. Considerations of protein and enzymic functional adaptations are also made to t r y and understand what some of the l i m i t i n g factors might be which would influence the type of adaptive mechanism for temperature compensations employed by a species; whether i t be by o v e r a l l protein restructuring, by isoenzymic induction, or by more subtle s p e c i f i c c e l l u l a r biochemical factors impinging upon protein c a t a l y t i c functions. 4 I I . MATERIALS 1. L i s t and sources of chemicals used Acrylamide Amberlite ion exchange resin MB-3 Amido Schwartze (Buffulo Black/Napthol Blue Black) Ammonium persulfate - reagent grade Bromopheno Blue Coumassie B r i l l i a n t Blue R Di t h i o t h r e i t o l Glycine (ammonia free) Hydrochloric acid ( H C 1 ) - reagent grade NN'Methylenebisacrylamide (bisacrylamide) Riboflavin (B 2) Sodium dodecyl sulfate (SDS) Trichloroacetic acid (TCA) N,N, N'N*-tetramethylethylendiamine (TEMED) Tr i t o n - x - 1 0 0 (octyl phenoxy polyethoxyethanol) Polyacrylamide gel electrophoresis apparatus Eastman Malinckrodt Eastman Mann Res. lab. Eastman Eastman Calbiochem A l l i e d Chem. Eastman Sigman Buchler Instr. Inc. 5 2. Experimental materials The eurythermal f i s h Gillichthyes m i r a b i l i s was used for the acclimation studies done at Scripps I n s t i t u t e of Oceanography, La J o l l a , C a l i f o r n i a . Tissues for trout studies were from frozen stored specimens that had been acclimated i n Vancouver by Dr. Peter Hochachka's laboratory at the University of B r i t i s h Columbia. One fresh trout from the San Diego area lake hatcheries was also used i n the preliminary studies of trout tissues. This trout had a thermal history of 24°-26°C. Since the source of Gillichthyes (from a lo c a l bait shop) was not controlled, size variation occurred with different batches of f i s h and genetic pools were likewise undetermined. Attempts were made at obtaining uniformity i n f i s h used for acclimation by selecting f i s h of simil a r size and from the same batch bought on a pa r t i c u l a r date. 6 I I I . METHODS 1. Acclimation. Gillichthyes m i r a b i l i s were cold acclimated at 8°C (6 hr l i g h t , 18 hr dark), and warm acclimated at 28°C (15 hr l i g h t , 9 hr dark). Controls were held at ambient sea water temperatures (20°-22°C). Fish were maintained i n 2-gallon glass j a r s , covered with metal screens to prevent f i s h from escaping especially during warm acclimation. For cold acclimation, f i s h i n ambient sea water were transferred to the cold room d i r e c t l y . Warm acclimation was done more gradually over a two day period to reduce the s i g n i f i c a n t l y higher death rate, p a r t i c u l a r l y during the f i r s t week of warm acclimation. To determine i f acclimation over a period of time would induce quantative s h i f t s i n protein patterns to become v i s i b l e q u a l i t a t i v e differences, studies were performed at intervals of 13, 20, 31, 38,45, and 52 days acclimation. One study of extreme condition of stress at 28°C for four months and starving for about two months p r i o r to the experiment, was made to determine what extent protein patterns would be affected by such extremes as compared to long term acclimation only. Fish were fed chopped frozen squid, also obtained from bait shops, and were transferred into clean temperature-equilibrated sea water once or twice a week. 7 2. Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis was employed for protein separations because this method gives good separations with repro-d u c i b i l i t y (Matson, 1965; Dehlinger § Schimke, 1971), and has the advantage of requiring m i c r o - l i t e r amounts of protein. The translucent gels could be stained to give bands that were easily photographed and from which densitometry readings were made. a. R e c r y s t a l l i z a t i o n of monomers (Loening, 1967). To ensure purity of acrylamide i n i t s monomeric form and to protect against ion contamination, both acrylamide and bis-acrylamide were r e c r y s t a l l i z e d . Both compounds are exceedingly poisonous and suitable precautions must be taken against contact with the skin or inhalation of the l i g h t c r y s t a l s . 70 grams of acrylamide were dissolved i n 1 l i t e r of chloroform at 50°C, f i l t e r e d hot without suction and allowed to r e c r y s t a l l i z e at -20°C, after which they were washed with cold chloroform and suction-dried. Bis-acrylamide was s i m i l a r l y p u r i f i e d using acetone (approximately 10 grams per l i t e r ) . Recrystallized material was stored under cool, dry, dark conditions to slow spontaneous polymerization and hydrolysis. Separate stock solutions of 15% (w/v) of acrylamide and suitable concentrations of bis-acrylamide i n P^ O can be stored for at least one month under ref r i g e r a t i o n and i n the dark. 8 b. Stock solutions (Davies, 1964) * Separation gel stock solutions (pH 8.9 ): (A) 1 N.HC1 *48.0 ml (B) acrylamide 28.0 gm t r i s 36.6 gm bis-acrylamide 0.735 gm TEMED 0.25 ml dH20 to 100 ml dH20 to 100 ml (C) Ammonium persulfate 0.28 gm/100 ml (10% w/v of acrylamide present) (made fresh on day of use.) * Stacking gel stock solutions (pH 6.7 ): (D) IN HC1 *48.0 ml (E) acrylamide 10.0 gm t r i s 5.98 gm bis-acrylamide 2.5 gm TEMED 0.46 ml dH20 to 100 ml dH20 to 100 ml (F) Riboflavin 4 mg/100 ml dH20 (G) Sucrose 40% (w/v) A l l solutions should be stored cold, and except for (G), stored dark. c. Working solutions and preparation of gels Working solutions: Stock solutions were allowed to warm to room temperature p r i o r to use. Separation gels (small pore) were made up with the above stocks at proportions of 2A:lB:ldH 20, and ammonium persulfate to give 10% w/v of acrylamide present. Stacking gels (large pore) were made with 1D:2E:F:4G. Chamber electrolyte buffer was made by a 1:10 d i l u t i o n of stock buffer (29 gm glycine, 6 gm t r i s , 5 ml N HC1, and 975 ml dH„0) to give 9 a f i n a l pH of 8.1. I t i s kept cold at the temperature of electrophoresis (4°C). Preparation of gels: The working solutions were degassed i n vacuo, for a few seconds p r i o r to use. Gels were made i n straight pyrex glass tubing, 80 mm x 5 mm (intern, diam.). Tubes for a set were cut from the same tubing to minimize variations i n e l e c t r i c a l resistance from column to column during electrophoresis. The glass tubes were acid cleaned, treated with a rinse of 1/200 parts of photo-flo, and dry before use. Treatment with photo-flo f a c i l i t a t e s gel removal from the tubes after electrophoresis. Similar result i s possible by the use of Plexiglass tubing (Davies, 1964). The tubes were capped and lined up perpendicularly for polymerization of the running gel solution, which was car e f u l l y pipetted into the columns to give a volume of 1.5 cm from the top after poly-merization. Slight shrinkage occurs during polymerization. The l a t t e r takes place i n the dark and requires about twenty to t h i r t y minutes. In order to obtain a f l a t sharp surface after polymerization upon which stacking gel w i l l follow, the running gel solution was immediately overlaid with a small amount of d i s t i l l e d water p r i o r to polymerization, by use of a fine needle micro-syringe. I t i s very important that surface i r r e g u l a r i t i e s be avoided as these affect migration of proteins into the running gel. S i m i l a r l y , care should be taken to avoid trapping a i r bubbles when running gel i s pipetted into the columns. I t has been suggested that polymerization should i d e a l l y be at the same temperature as that for electrophoresis to eliminate thermal contraction or expansion of the gel matrix (Chrambach § Rodbard, 1971), Preparation of the stacking gel required s i m i l a r precautions for getting uniformity of gel surface and polymerization. The water 10 above the running gel was removed and residual water soaked up with f i l t e r paper. Stacking gel was about 6 cm high after polymerization. For good stacking effect the volume should be greater than 50% of the protein solution volume put on the column for electrophoresis. After being overlaid with water, stacking gel polymerization was i n i t i a t e d by the presence of l i g h t by l i n i n g the columns at an uniform distance of about two inches from a day-light flourescent tube. Polymerization takes about ten to f i f t e e n minutes, causing the stacking gel solution to become uniformly opaque when polymerized. After polymerization, gel tubes were removed from t h e i r caps and inserted uniformly into the electrophoresis apparatus, making sure no a i r bubbles were trapped, and leaving s u f f i c i e n t column length to allow for observation of protein stacking and migration into the running gels during i n i t i a l electrophoresis. A few drops of the tracking dye Bromophenol Blue was mixed into the upper buffer compartment, and after temperature of the gels were equilibrated to the 4°C conditions of electrophoresis, protein samples (made heavy by 40% sucrose) were micro-syringed onto the stacking gel for electrophoresis. In s p l i t - g e l s , a snugly f i t t i n g piece of index card was inserted into the tube and s l i g h t l y into the stacking gel p r i o r to protein application, and electrophoresis begun immediately after protein samples were applied to the separated sides of the tube (Dunker § Rueckert, 1969). Electrophoresis was carried out with an i n i t i a l current of 2 ma per tube. After protein entered the separation g e l , current was increased to 5 ma per tube. Current should not exceed 5 ma per tube to avoid excessive ohmic heating, which results i n protein migration pattern a r t i f a c t s . I n i t i a l low current i s a precaution against 11 convective losses of the sample into the upper reservoir (Davies, 1964). Electrophoresis was terminated when the tracking dye was about 3 mm from the bottom of the gel (in about 1.5 hrs). A variable voltage regulated power supply was used (Heath K i t Inc.). 3. Sodium dodecyl sulfate gels Proteins of mitochondria, post mitochondrial supernatants, and microsomal fractions were also studied using SDS polyacrylamide gels, which separates proteins based upon th e i r size differences alone and not on charge. See appendix A for further details of the principles operating i n th i s system. Electrophoresis techniques were si m i l a r to those described for polyacrylamide gels, and were adapted from the methods of Weber and Osborn (1969), and from Kiehn and Holland (1970). 7% gels were prepared without stacking gels, using the solutions given for polyacrylamide gels, but with the addition of 0.1% SDS, and 0.1% 2-Me0H and 15 mg/ml ammonium persulfate added immediately before use. Electrophoresis chamber buffers likewise contained an addition of 0.1% SDS. Tissue homogenated for SDS electrophoresis were treated with 0.1% SDS and 2-MeOH, and incubated at 37°C for t h i r t y minutes, then made heavy with 40% sucrose and mixed with about 5 u l of tracking dye (0.05% Bromophenol Blue i n water). D i a l y s i s , which i s often recommended, was omitted as i n Weber and Osborn. Mitochondria were freeze-thawed before the SDS mercaptoethanol treatment to disrupt the p a r t i c a l s . Electrophoresis at 8 ma per tube took about 3-4 hours for the 8 cm gels used. The gels were then stained with Coumassie Blue as described i n section 4 of the methods. 12 4. Staining and recording of protein bands i n SDS and polyacrylamide  gels Amido Schwartze and Coumassie Blue dyes were used. Comparisons of the dyes showed better resolution of minor protein bands by Coumassie Blue, which was the dye u t i l i z e d i n the majority of the experiments. Staining procedures were modified from Chrambach et a l . (1967), and destaining procedures were from those used by Weber and Osborn (1969). a. TCA f i x a t i o n of proteins Gels were removed from t h e i r tubing for protein f i x a t i o n and staining by careful rimming under water with a long fine blunt needle syringe and by s l i g h t pressure of pipette bulb. The gels were immediately put into test tubes containing 12-15% t r i c h l o r o a c e t i c acid (TCA) for f i x a t i o n of the protein bands separated during electrophoresis. Maximum protein band sharpness i s obtained by quick f i x a t i o n at these somewhat higher concentrations of TCA than the 10% normally used (Chrambach e_t a l . , 1967). b. Staining of proteins Staining was done separately from protein f i x a t i o n since the dye i s somewhat insoluble i n TCA, and especially with the higher concentrations u t i l i z e d i n these methods. The staining solution was made up i n 454 ml 50% MeOH, 46 ml acetic acid (9%), and 0.125 g Coumassie Blue (0.025% f i n a l c one). A stock solution of 2% Coumassie i n 50% MeOH i s stable. Staining time was 1-2 hours or longer, depending on the i n t e n s i t y of sta i n required. c. Destaining and storage This was accomplished by soaking gels i n washes of the destaining solution which was made of 7% acetic acid and 5% methanol. Gels were stored i n 7% acetic acid and i n the dark to prevent fading. No v i s i b l e shrinkage of gels was found when stored t h i s way, as compared to storage i n TCA, which i s often recommended. d. Photography and densitometry graphs of gels Gels were put i n a clear p l a s t i c dish, covered with d i s t i l l e d water and photographed by transmitted l i g h t using Polaroid black and white 4 x 5 55P/N type f i l m . Increased photographic s e n s i t i v i t y to protein stained bands was obtained by use of a dark green f i l t e r (No. 74). Red f i l t e r caused dense blue stained bands to become too black and l i g h t e r bands to be cut off. Timing was varied for optimal exposure at f32. Negatives were prepared for p r i n t i n g by treatment i n 18% sodium s u l f i t e as directed from Kodak, and rinsed i n photo-flo. Densitometry graphs of the negatives were made from a double beam recording microdensitometer, MK IIIB from Joyce, Loebl and Co.. Recordings were also done d i r e c t l y from the gels for comparison. Absorption spectrum studies of the gels were done at 325 mu for Amido Schwartze and at 560 mu for Coumassie Blue. Results from these methods were comparable and the densitometer method was selected for i t s convenience. Radioactive labeled nuclear proteins separated with sodium dodecyl sulfate polyacrylamide gels have been shown to give 32 densitometry readings that correlate with P r a d i o a c t i v i t y for gel s l i c e s (Johnson, et_ al_., 1974). 5. Starch gel electrophoresis Starch gel electrophoresis was selected for studies of s p e c i f i c enzymes because the gel slab could be stained i n one step. A system of discontinous buffers was used which reduces electrophoresis time (Poulik, 1957), and therefore d i f f u s i o n ; factors that contribute to better protein separations. Buffer for the starch gels contained 0.076 M t r i s and 0.005 M c i t r i c acid (pH 8.6), and buffer for electrode chambers contained 0.3 M boric acid and 0.05 M sodium hydroxide. Electrophoresis was followed by the v i s i b l e brown colored boundary which increased i n intensity as the boundary moved along the gel towards the cathode. This boundary i s probably caused by the replace-ment of the c i t r a t e ions i n the gel by borate ions from the electrode vessels according to Poulik ( i b i d . , 1957). The migration of proteins i n this system gave good results i n 3-4 hours using a potential gradient of 6 V/cm and at 4°C. The basic procedures for starch gels followed the method introduced by Smithies (1955). P a r t i a l l y hydrolyzed starch obtained commercially was made up i n appropriate buffer to make a 10% gel, using an erlenmeyer flask. The starch solution was heated slowly with continuous s w i r l i n g and brought to the b o i l i n g point for 1-2 seconds. The hot solution was degassed and poured into the electrophoretic tray, covered securely with a sheet of heavy plexiglass making sure no a i r bubbles were caught between, and l e f t to s o l i d i f y at room temperature. After careful removal of the glass plate the gel was covered at a l l times with seran wrap to maintain stable moisture content. The gel was cooled to 4°C before protein was applied for electrophoresis. This was done by saturating pieces of Whatman f i l t e r paper with the protein solution and then inserting these into s l i t s cut perpendicularly to the gel at the o r i g i n and set a few centimeters i n from the end of the gel. Contact between e l e c t r o l y t i c vessels and the gel was made by several sheets of f i l t e r paper saturated with electrode buffer. 15 b. Specific enzymes studied A preliminary study was made of some regulatory enzymes i n Gill i c h t h y e s. A more comprehensive l i s t of enzymes have subsequently been studied by Dr. G. Somero, which provided s i m i l a r indications. This l i s t i s given i n table I, and was graciously supplied by Dr. Somero, to whom I am greatly indebted. TABLE I. PROTEINS EXAMINED ELECTROPHORETICALLY Dehydrogenases: ©<-glycerophosphate dehydrogenase ethanol dehydrogenase glucose-6-phosphate dehydrogenas e i s o c i t r a t e dehydrogenase lactate dehydrogenase malate dehydrogenase octanol dehydrogenase 6-phosphogluconate dehydrogenase xanthine dehydrogenase Other: acid phosphatase esterases fumarase glutamate-oxaloacetate transaminase leucine aminopeptidase peptidases phosphoglucomutase phosphohexose isomerase general protein * grateful thanks are given to Dr. George Somero for this table 17 6. Tissue preparation and c e l l u l a r fractionation a. Tissue preparation Fish were k i l l e d by decapitation. Liver and brain tissues were quickly excised and homgenized each i n 5 volumes of cold TMK-S (0.25 M sucrose, 0.05 M t r i s - H C l , pH 7.6, 0.025 M KC1, and 0.005 M MgCl^, based upon the methods of Haschemeyer (1967), and with modifications from Schnaitman (1969), and Kiehn and Holland (1970) for tissue preparations for SDS gel electrophoresis. A l l procedures were done on ice and centrifugation was at 2°-4°C. In studies comparing individual variations of animals from the different acclimation temperatures, tissues from each animal were homogenized separately. Attempt at uniformity i n a l l procedures was made to minimize variations due to mechanical manipulations. Brain tissue homogenization was accomplished by 10 strokes of a close f i t t i n g Dounce B homogenizer. Liver tissue was minced and homogenized using 5 strokes of the loose f i t t i n g Dounce A homogenizer, f i l t e r e d through a nylon mesh to remove connective tissue, then homogenized with 15-20 strokes of the Dounce B homogenizer. Muscle tissue was homogenized i n 10 volumes of d i s t i l l e d water for i s o l a t i o n of glycolysis proteins only (White, Handler, § Smith, 1964). Muscle tissue was obtained by removal of the epaxial muscle mass and stripping i t of skin, mincing i t f i n e l y with a Waring blendor and then f i l t e r i n g with nylon mesh. The f i l t r a t e was homogenized with a ground glass homogenizer, centrifuged b r i e f l y at low speed to remove f i b r i l l a r material and the supernatant homogenized with the Dounce B homogenizer. b. Subcellular fractionation Subcellular fractionation followed the scheme shown below, which was modified from the composite flow chart i n Mahlar and Cordes (1966), and from the authors cited i n the preceeding section for tissue preparation: Homogenize minsed tissue (0.2g/ml) i n TMK-S, with Dounce A, f i l t e r , Dounce B 400 x g, 10 min (twice) 1 RBC 750 x g, 10 unbroken c e l l s (twice) connective tissue 1 crude nuclei large membrane fragments post nuclear supernatant 7,700 x g, 15 min (twice) 1 crude mitochondria post mitochondrial (+ 2% t r i t o n -supernatant I x 0.005 M (PMS I) DTT for electro-phoresis) 14,800 x g, 30 min low speed microsomal fraction rough and smooth vesicles membrane fragments (treatment with SDS MeOH for electrophoresis) PMS II (+DTT for electrophor.) + 2% t r i t o n - x 144,000 x g, 2hr 1 soluble insoluble (+ DTT for electro-phoresis) 105,000 x g, 90 min ribosomal p e l l e t 19 The fat layer that forms at the meniscus should be removed after centrifugation, otherwise l i p i d interference with clean fractionation occurs. This i s especially important i n polyribosomal i s o l a t i o n (Haschemeyer, 1967) . The i n i t i a l centrifugation at 400 g was omitted i n studies that did not include nuclei i s o l a t i o n . Likewise, the 750 g centrifugation step was omitted i n studies limited to post mitochondrial fractions. The homogenates were then d i r e c t l y centrifuged at 7,700 g following the scheme shown. Crude nuclei and mitochondrial p e l l e t s were made more pure by three washes with tris-sucrose (0.05M t r i s , pH 6.7, 0.25M sucrose). The washed p e l l e t s were then resuspended i n 20 x volume of tris-sucrose and centrifuged at 14,000 g for 20 minutes. The p a r t i c l e s were resuspended i n TMK-S containing 2% triton-x-100 and 0.005 M d i t h i o t h r e i t o l (DTT) for acrylamide electrophoresis. Freeze thawing was employed to promote l y s i s . In sodium dodecyl sulfate acrylamide gel electrophoresis, treatment with SDS and mercaptoethanol and incubation at 37°C was performed p r i o r to electrophoresis, as described i n section 3 of the methods. Post mitochondrial supernatant was treated with triton-x-100 and DTT for electrophoresis, or alt e r n a t i v e l y treated with 2% t r i t o n -x-100 only and centrifuged at high speed to give t r i t o n - x soluble and insoluble fractions for electrophoresis. Microsomal fractions were isolated by centrifugation of the untreated post mitochondrial supernatant (PMS I) at 14,800 for 30 minutes. The supernatant from t h i s was layered over 40% sucrose made up i n t r i s , and centrifuged at 105,000 g for 90 minutes to give a ribosomal p e l l e t which was used for urea gel electrophoresis (Haschemeyer, 1967). c. Polyribosomal i s o l a t i o n (Haschemeyer, 1967; Ling & Dixon, 1970). Polyribosomal i s o l a t i o n was obtained by homogenation of l i v e r i n 3 volumes of TMK-S pH 7.6, using 3 strokes of Dounce A, f i l t e r i n g with nylon mesh, and homogenating gently with 5 strokes of the Dounce B homogenizer. The homogenate was spun at 7,700 g for 20 minutes and the post mitochondrial supernatant treated with 2% triton-x-100 for 10 minutes. The PMS was then divided into two fractions; one for control was treated with 2 ug/ml ribonuclease. Equal volumes of control, PMS, and TMK-S with 2% t r i t o n - x were carefully layered onto three l i n e a r sucrose gradients (10-35% w/v at 4°C) containing 0.01 M tri s - H C l pH 7.6, 0.02 M KC1 and 0.005 M MgCl 2- Centrifugation was done i n SW25 orSW27 Spinco rotors at 25,000 or 26,000 rpm for 0.5 to 2 hours. The centrifuged gradients were punctured at the bottom with a fine needle and equal volume fractions were collected for analysis of absorbance at 260 urn. A l l procedures p r i o r to this l a s t step must be done on ice. To protect against RNase contamination, a l l apparatus for homogenation was acid washed, and handling of apparatus parts used i n direct contact with the tissue homogenate was avoided. Ribonuclease-free sucrose was prepared by b o i l i n g sucrose i n a double b o i l e r for one hour. •IV. RESULTS 1. Acclimation The results presented here are selected from three batches of f i s h that were acclimated over the periods July 2 through October 27. (See chart below.) During t h i s time sea water controls experienced varying ambient temperatures as shown here and i n figure 1. ACCLIMATION DAYS DAY OF FISH AMBIENT SEA WATER CONTROLS (8 and 28 C) EXPERIMENT BATCH mean °C range °C 13 7/16 I 20 18-22 20 8/2 II 22 19-24 31 7/3 I 22 19-24 38 9/24 I I I 19 16-21 45 8/16 II 22 19-24 52 10/27 II 17 14-19 Fish survival during cold acclimation was s i g n i f i c a n t l y better than i n warm acclimation, especially during the i n i t i a l period of acclimation. The precaution for gradual acclimation described i n the methods reduced this difference. Fish i n cold acclimation maintained good appetite although t h e i r food consumption decreased compared to warm f i s h . Swimming a c t i v i t y i n cold acclimation f i s h was also reduced and the f i s h tended to remain at the bottom of t h e i r j a r s . Warm acclimation f i s h were more active and tended to try to escape from t h e i r containers; they also exhibited surface a i r gulping behavior. (See discussion V-2.) Body weight changes over the acclimation periods were also different under the two acclimation temperatures. In general, cold 22 Fig. 1. Surface sea water temperatures--1971. From o f f the p i e r at Scripps I n s t i t u t e of Oceanography, University of C a l i f o r n i a , La J o l l a : Temperatures experienced by ambient sea water controls i n the studies of Gillichthyes m., as indicated i n the text on page 21. acclimation f i s h tissues had greater fat content; t h e i r l i v e r s were especially high i n l i p i d s and were large and pale. Warm acclimation f i s h tended to lose weight over long term holding at 28°C. The decrease i n protein s t a i n obtained i n polyacrylamide gel electrophoresis may be due to this loss of weight being reflected at the tissue biochemistry level as discussed i n section V. Livers from long term acclimated f i s h were dark and tended to atrophy after about s i x weeks. Starvation at 28° C accentuated these abnormalities. 2. Gel electrophoresis Results from acrylamide gel electrophoresis for Gillichthyes are shown i n figures 2 to 13, and for trout i n figures 14 to 15. Graphs for some of these figures are to be found i n appendex B i f more careful comparisons are wanted i n reference to the differences given i n the following results. Acclimation effects on Gillichthyes were greater i n l i v e r tissue than i n muscle or brain. Muscle (figure 2) and brain (figure 3) both showed no apparent changes i n protein pattern d i s t r i b u t i o n using acrylamide gel electrophoresis. Liver tissue was studied i n greater d e t a i l . Preliminary studies were made to determine the effects that freeze storage of tissue would have on electrophoresis r e s u l t s . Proteins were stable with overnight freeze storage but longer storage increased denaturation of the slower migration species, affecting the resolution of minor protein bands with one week storage (figure 4b vs. 4a), and producing greater smearing and aggregation effects with storage over months (figure 5). I t was found that starvation at high acclimation temperature changed the d i s t r i b u t i o n pattern of proteins more than short term freeze storage, but that resolution of the protein bands were good; 24 l i v e r tissues showed a greater number of fast migration protein bands around band 13 and 14, and a greatly increased peak for band 18 (figure 6). Starvation and high temperature conditions also caused, major protein pattern changes i n muscle tissue (figure 1 vs. 6; graph 1 vs. 5). Acrylamide gel electrophoresis of l i v e r post mitichondrial supernatant (7,700 x g, 20 mins) i n general produced 20 c l e a r l y v i s i b l e protein bands.' 13 days acclimation (figure 7a; graph 7). No s i g n i f i c a n t differences i n protein d i s t r i b u t i o n patterns were observed for short term acclimation of 13 days. The s l i g h t reduction i n the intensity of the faster migration bands 18-20 i n warm acclimation may be due to d i l u t i o n effects from a s l i g h t l y faster lead i n migration rate. A discussion of factors affecting migration rates i n gel electrophoresis is given i n appendix A. The resolution of bands 3, 4, and 5 was better i n cold than i n controls, of warm f i s h . These results were duplicated i n repeat electrophoretic runs of the same tissue homogenates (figure 4a; graph 2). 20 days acclimation (figure 7b; graph 7). A longer electropho-r e t i c run was performed i n this experiment to attempt better separation of the slower moving proteins. There was again a somewhat more intense staining of bands 3, 4, and 5 from 4°C f i s h , r e l a t i v e to 28°C or sea water control f i s h . Since the s i m i l a r results were obtained from f i s h of two different batches, these minor variations between acclimation temperatures may be s i g n i f i c a n t differences. 31 days acclimation (figure 8a; graph 8). Results from this experiment were poor, but within the range of resolution good rep r o d u c i b i l i t y of protein banding pattern was obtained with duplicate gels. No apparent differences between 8°C and sea water controls were 25 indicated. 58 days acclimation (figure 8b; graph 9). Batch I I I f i s h used here exhibited greater number of protein bands, and these bands contained many variations i n the regions of protein bands 3-13. Variations between tissues from individual f i s h were as great as between f i s h from different acclimation temperatures. This batch of f i s h exhibited a s i m i l a r range of variations i n a 20 days acclimation electrophoresis study. Possible polymorphic contributions to these variations are discussed i n section V. 45 days and 52 days acclimation (figure 9, graph 10; figure 10). Warm acclimation tissues i n general appeared to have poorer protein resolution i n the slower migration species (bands 3, 4, and 5), compared to cold to controls, and poorer separation of some bands (7, 8) compared to controls. It might be that these apparent differences are accentua-tions of the s l i g h t v a r i a t i o n indicated e a r l i e r for 13 and 20 day acclimations. Miscellaneous studies Comparisons of low speed and high speed supernatant proteins (figure 11a) suggests that sea water controls have somewhat higher concentrations of slower migration larger protein species i n the slow speed supernatant. The reverse, a reduction of larger slower migration protein species, may be indicated for warm acclimation. More careful quantitative studies would have to be done to support these preliminary indications. Mitochondria trition-x-100 soluble proteins (figure l i b ) revealed differences i n protein pattern which were also apparent i n s p l i t gels. However, i n s u f f i c i e n t study was made i n order to conclude i f the differences were re a l or were due to individual f i s h v ariations, as was found i n the l i v e r tissues of batch I I I f i s h . Acid gel electrophoresis of various subcellular fractions (figure 12) gave poor resolution and basic gels were therefore u t i l i z e d for the studies given. A discussion of factors necessary for good electrophoretic resolution, which have to be worked out for each system of proteins under study, i s given i n appendix A. Mitochondrial SDS (sodium dodecyl sulfate) acrylamide gel electrophoresis results were very s i m i l a r for a l l temperatures, with no apparent differences revealed by s p l i t gels (figures 13a, 13b; graph 11). Variations i n the number of minor bands i n the upper portions of the gels ('a' on graph) may be due to concentration effects and other factors affecting this upper region of the gel as discussed i n appendix A. Trout tissues Long term storage of trout l i v e r s rendered them useless for electrophoretic study, as seen by the great loss i n protein band resolutions (figure 15) r e l a t i v e to results from fresh tissues (figure 14). The greater number of protein bands found i n the intermediate temperature (12°C) muscle was more s i m i l a r to the fresh 28°C trout tissue (figure 14) than to stored 4° or 17°C tissues. This could suggest that acclimation conditions may affect the nature of protein structural s t a b i l i t y and especially of the larger slower migration species, which s i m i l a r l y appeared to be more l a b i l e i n warm acclimated Gillichthyes l i v e r proteins. 27 Acrylamide gel electrophoresis results: Fig. 2 to 13, Gillichthyes mirabilis. Fig. 13 to 15, trout. Abbreviations: AS = Amido Black general protein dye. b = bromopheno blue tracking dye. BSA = bovine serum albumin. CBB = Coumassie B r i l l i a n t Blue general protein stain. C = control, held at ambient sea water temperatures. Numbers on side of columns refer to protein bands. For comparison with densitometry graphs, see Appendix B, for those gels numbered with brackets around their numbers. Acclimation temperatures: 8°C, cold; 28°C, warm; controls, as indicated or see table on page 21. Proteins: Liver and brain tissues were prepared for electrophoresis ' by homogenization in 5 volumes of cold TMK-S (0.25 M sucrose, 0.05 M Tris-Hcl, pH 7.6, 0.025 M KC1, 0.005 M MgCl^ using the dounce B homogenizer as described in the methods (p.17). Proteins shown in a l l figures, except where indicated, are •from post mitochondrial fractions, (7.,700;x; giir20tmin.)i^treated with 2% triton-x-100 and 0.005% dithiothretol for electrophoresis. Mitochondrial fractions were washed with 0.05M sucrose and 0.05M tris-HCl, pH 7.6 and treated with 0.1% SDS and 0.1% mercaptoethanol at 37°C for 30 min, then made heavy with 40% sucrose and tracking dye added, prior to electrophoresis (methods, p. 11) Fig. 2. Gillichthyes m. muscle: 13 days acclimation, glycolysis proteins fro- water soluble, 17,000 g, 20 min. supernatant. Duplicate gels for each temperature are from pooled tissues of two or three f i s h . 27a 28 Fig. 3. Gillichythes m. brain: (a) 21 days acclimation. Intensity differences are due to 50x, 100\, 150x, of proteins applied to columns. Gels for each temperatures are from pooled tissues of 2 f i s h . Gel 1-3, 8°C; gels 4-6, 22°C; gels 8-10, 28°C. (b) 45 days acclimation. AS, amido black dye duplicates of gels 1-6. Gels for each temperature are duplicates using the same tissue homogenate. 28a 29 Fig. 4. Liver proteins short term freeze storage. Tissues quick frozen i n dry ice - ethanol. See Appendix B, graph I I , I I I . (a) Fresh tissue; 13 days acclimation. Compare with f i g . 7a; overnight freeze - storage. 8°C, gel 1,2; 19°-22°C, gels 3, 4; 28°C, gels 5, 6. (b) Frozen 1 week; same tissue homogenates from (a). 29a 30 Fig. 5. Liver proteins long term freeze storage. Gels 1,2, fresh ambient sea water f i s h . Gels 4-6, tissues frozejx.for. months. 30 a 31 Fig. 6. Starved stressed 28°C Gillichthyes m. tissues. Duplicate gels 1,2 = l i v e r ; 3,4 = muscle; 5,6 = brain. Gels 7,8,9, same tissues but gels dyed with amido black. See Appendix B, graph IV, V, VI, respectively for l i v e r , muscle, brain. 31 a 32 Fig. 7. (a) 13 days acclimation 8°C, gels 1, 2; 19°-20°C, gels 3,4; 28°C, gels 5,6. (See Sppendix B, graph VII). (b) 20 days acclimation 8°C, gels 1,2; 21°-24°C, gels 3,4; 28°C, gels 5,6. S p l i t gel 8°/28°C, gel 7. (See Appendix, graph VI I I ) . 35 Fig. 8. (a) 31 days acclimation 8°C, duplicate gels 1,2 and 3,4; controls, 19°-24°C, : gel 5, and duplicate gels 6,7. (See Appendix, graph IX). (b) 38 days acclimation 8°C, duplicate gels 1,2 and 3,4; controls, 16°-21°C, duplicate gels 5,6 and 7,8; 28°C, duplicate gels 9,10 and 11,12. (Appendix B, graph X). 33a 34 Fig. 9. 45 days acclimation 8°C, duplicate gels 1,2; controls ambient sea water 16°-21°C, duplicate gels 3,4; 28°C, duplicate gels 5,6. Amido black stained gels, repeats of the some sequence. (Appendix B, graph XI). 34 a 35 Fig. 10 52 days acclimation Each gel i s from separate f i s h : 28°C, gels 1,2,3; 8°C, gels 4,5,6; controls, gels 7,8. S p l i t gels 28°/8°C. 35 a / 36 Fig. 11. (a) Low speed v.s. high speed post mitochondrial supernatants Basic gels: Low speed, 28°C, gels 1,2; controls, gels 3,4. Acid gels: Low speed, 28°C, gels 5,6; controls, gels 7,8. High speed, 28°C, gels 9,10; controls, gels 11,12. (b) Mitochondria triton-x-100 soluble proteins. Acid gels. 36 a Pig- 12 Acid gels of l i v e r tissue sub-cellular fractions. 37a 38 Fig. 13. Sodium dodecyl sulfate acrylamide gels of mitochondria SDS, 2-mercaptoethanol soluble proteins: (a) T r i p l i c a t e gels: 8°C; ambient sea water. Duplicate gels: 28°C. S p l i t gels, 8°/28°C. (Appendix B, graph XII for gels 3, 5, 8) . (b) Duplicate gels of each temperatures fstained with Coumassie B r i l l i a n t Blue and Amido Black dyes. Gel 1, bovin serum albumin. 38a 39 Fig- 14. Fresh 24°C trout tissues: l e f t to right- brain, l i v e r , muscle. Stained with Coumassie B r i l l i a n t Blue, gels 1-3, and with Amido Black, gels 4-6. 39a Fig. 15. Frozen trout tissues: (a) Liver. Each gel from separate f i s h . Two gels each for 4°C, 12°C, and 17°C acclimations. Stained with CBB, gels 1-6, and with AS, gels 7-12. (b) Muscle: Same as for l i v e r for acclimation temperatures Stained with CBB, gels 1-4, and with AS, gels 5-8. 40 a 41 3. Polyribosome and monoribosome p r o f i l e Results from the sucrose density gradients (10-35%) did not indicate c l e a r l y any s h i f t i n the polyribosome and monoribosome p r o f i l e for studies of 2 weeks acclimation i n Gillichthyes m i r a b i l i s . 42 V. DISCUSSION 1. Electrophoretic results and tissue comparisons Polyacrylamide gel electrophoresis separates proteins based upon t h e i r charge and size differences. A discussion of principles involved i n polyacrylamide gel electrophoresis and the conditions that contribute to optimal results i s given i n appendix A. The results from the present studies indicate remarkable rep r o d u c i b i l i t y of protein d i s t r i b u t i o n patterns between f i s h from a l l acclimation temperatures. The minor variations i n band separation i n the regions of bands 3, 4, and 5 were quantitative rather than q u a l i t a t i v e and may r e f l e c t phenomena i n t r i n s i c to the gel near the o r i g i n , a phenomena also observed by other investigators and c a l l e d a "haze zone" by Matson (1965). However, since the s l i g h t variations were reproducible, and where most apparent, they may r e f l e c t changes induced during warm acclimation. Factors that i n t e r f e r e with good resolution and cause variations can be due to mechanical i r r e g u l a r i t i e s produced at the stacking gel and running gel interphase, or due to protein aggregation and plugging of the gel interphase by larger insoluble proteins. Mechanical factors i n these experiments were minimal. There was a problem of protein aggregation and plugging of the gel, which affected the r e l a t i v e migration rates and reduced resolution of minor proteins, as observed i n the 31 days acclimation study. Plugging of gel pores at the upper gel interphase can interfere with even electrophoretic f i e l d and cause joule heating and protein streaking a r t i f a c t s . To overcome this problem, high speed centrifugation and SDS and mercaptoethanol treat-ments of homogenates and gels were used. High speed centrifugation 43 reduced the number of proteins and the i n t e n s i t i e s of the protein bands obtained. Triton-x-100 treatment greatly improved post mitochondrial supernatant r e s u l t s , but was not s u f f i c i e n t for s o l u b i l i z a t i o n of membrane proteins. Triton-x treatment of mitochondrial and microsomal fractions i n rat l i v e r studies (Dehlinger and Schimke, 1971) indicated that 50% of rat l i v e r membrane proteins were s o l u b i l i z e d and that these had molecular weights greater than 50,000; t r i t o n - x insoluble proteins were heteroge-neous i n si z e . SDS and mercaptoethanol treatment and electrophoresis with SDS gels produced good protein band resolutions with no observable protein plugging of gel surface. Two points should be kept i n mind when considering the protein bands d i s t r i b u t i o n s : (1) Before a band can be considered a true single unit protein, correlative studies must be performed with known proteins. Single chains of serum albumin have been shown to aggregate and give multiple zones i n acid gels (Poulik, 1966). (2) Since the amount of dye that binds to a protein varies with proteins, densitometry graphs provide only r e l a t i v e quantitative comparisons unless reference quantities of the proteins have been electrophoresed at the same time. Comparisons of r e l a t i v e quantities of proteins from the same experiment should therefore be more r e l i a b l e than between graphs from separate runs. The present studies would seem to indicate that thermal .acclimation induces no apparent major changes i n the proteins of Gillichthyes m i r a b i l i s for the tissues investigated as described. Longer term acclimation may affect minor protein species found i n the regions of slower migration larger sized proteins. Longer term acclimation may also have overall effects on protein band concentrations by either influencing structur al properties of the proteins, s i m i l a r to those produced by freeze storage (which likewise produced loss of 44 resolution), or by decreasing the pool of tissue proteins. The protein patterns that were obtained from warm stressed starved f i s h indicated d e f i n i t e d i s t r i b u t i o n changes and therefore greater restructuring of proteins. These effects were i n the faster migration protein species. The observed decrease i n proteins from l i v e r tissue of f i s h subjected to prolonged warm acclimation without starvation may therefore be r e f l e c t i v e of depletion of protein pools. This would suggest that warm acclimation at 28°C imposed greater stresses for Gillichthyes m. than did cold acclimation. This was also apparent by the gross observations of weight loss and l i v e r atrophy over prolonged holding at the elevated temperature. Questions arise from these for considering how the capacity to adapt metabolically may correlate with the eurythermic nature of th i s species. What are some of the l i m i t s set by the thermal environments encountered i n the ecology of th i s species, and what might be some of the mechanisms that enable i t to become functionally adapted to eurythermic conditions? 2. Acclimation adaptations Although no overall major restructuring of proteins were observed by gel electrophoresis, the gross metabolic and physiological adjustments observed (changes i n l i p i d content, physiological a c t i v i t i e s , and i n the different adaptive capacity to cold and warm stress) would indicate that induction of metabolic processes do occur i n response to temperature. Early experiments by Summer and Doudoroff (1935) showed that Gillichthyes exposed to warm and cold for as l i t t l e as 30 minutes became more resistant to subsequent l e t h a l temperatures. The resistance increased at diminishing rates over 30 minutes to ten days exposures. It was found that previous warm acclimation provided better resistance to warm than did previous cold acclimation to cold stress. I t would 45 seem then that whatever the adaptive processes evolved by this eurythermic species, the capacity for heat adaptation could be a more important phenomena, a point that may be especially relevant to the ecology of t h i s species. Seasonal variations of i n t e r t i d a l pool temperatures have been shown to range from 12°C to 34°C, while surface sea water ranged from 14°C to 22°C for the year monitored (Norris, 1963). (See f i g . 1; surface water temperatures for this study.) The burrowing behavior of G i l l i c h -t h y e s and t h e i r a b i l i t y for buccal resp i r a t i o n would also indicate that this species has evolved the capacity to adjust to conditions of heating and low oxygen tensions of i n t e r t i d a l ecology. The prolonged stress at 28°C i n the present studies would be towards the upper l i m i t s of tolerance for G i l l i c h t h y e s , and therefore might account for the observed variations that were accentuated with prolonged holding at t h i s tempera-ture. In nature, Gillichthyes experience short term diurnal heating, and these fluctuations would induce prolonged resistance adaptations. It has been reported that f i s h can respond to as l i t t l e as 0.5°C temperature changes (in Fry § Hochachka, 1971). In G i l l i c h t h y e s , behavioral selection may lessen the degree of temperature changes encountered i n diurnal temperature fluctuations. However, the i n t e r t i d a l ecology would s t i l l subject the f i s h to thermal ranges far greater and quicker than those encountered by seasonal adaptors. Temperature, then, has to be translated into more immediate physiological parameters. 3. Protein and enzymic functional adaptations The p o s s i b i l i t y that eurythermic capacity was derived from thermally induced changes i n protein synthesis rates, or changes i n enzymic species during acclimation was shown to be negative, from the 46 present studies of polysomes and monosomes, and from the extensive l i s t of enzymes investigated (table I ) . The question of thermally induced changes i n proteins during acclimation has also been studied i n G i l l i c h - thyes from the point of protein degradation (Somero § Doyle, 1973). I t was found that protein degradation rates were different i n different tissues of warm and cold acclimated f i s h . A l l these findings lead to the question: What are the i n t r i n s i c q u a l i t a t i v e s t r u c t u r a l properties of enzymes from eurythermal f i s h that give the animal i t s biochemical f l e x i b i l i t y under varying thermal conditions. Also, how might temperature affect enzyme function by influencing l o c a l c e l l u l a r environments, and therefore enzymic a c t i v i t i e s . On the one hand, simple models i n nature have often been held to be the rule, and so i n some cases of poikilotherms, adaptive compensa-tion to thermal changes may be by restructuring of certain regulatory enzymes (Hochachka, 1968, Hochachka § Lewis, 1970). On the other hand, c e l l u l a r metabolism consists of complex integrated processes. These processes have to be orchestrated to a l l the f i n e l y changing nuances that enable the organism to achieve delicate balances, the summation of which encompasses physiological v i a b i l i t y . Temperature as only one modulatory force might therefore affect metabolism by influencing molecular processes on overall physiological levels. Various ideas have been suggested that focus upon possible i n t r i n s i c q u a l i t a t i v e differences i n the nature of proteins that may account for t h e i r biochemical adaptability. One view suggests that gene duplication has provided means for s l i g h t variations i n functional proteins and that these s l i g h t differences were p r e f e r e n t i a l l y exploited during ontogeny (Ohno et_ al_. , 1967). Widespread natural v a r i a t i o n of 47 many proteins have been amply reported (Bailey e_t al_., 1969; Dando, 1974; Hattingh, 1973; Johnson, 1971). The greater degree of protein pattern variations observed i n the f i s h from batch I I I i n the present investigation might be r e f l e c t i v e of increased polymorphic expressions induced by ecological parameters experienced by that batch of f i s h i n t h e i r developmental history. Other studies comparing eurythermic and stenothermic species have indicated differences i n enzymes, r e f l e c t i v e of t h e i r b i o l o g i c a l thermal niche. In one study, eurythermic rainbow and brook trouts, which experience seasonal temperature changes, exhibited heterogeneity i n the multiple forms of soluble i s o c i t r a t e dehydrogenases. Stenothermic lake trout, which experiences very stable temperature, exhibited only one subunit form of the enzyme (Moon 5 Hochachka, 1971). Comparisons of the capacity for acclimation to eurythermic or stenothermic conditions i n two closely related species have indicated that the g l y c o l y t i c enzymes, lactate dehydrogenase and pyruvate kinase, exhibited d i f f e r e n t a b i l i t i e s to acclimate, r e f l e c t i v e of t h e i r species natural habitat (Wilson et a l . , 1974). Tissue s p e c i f i c enzymic a c t i v i t y differences for an enzyme from homeothermic and heterothermic tissues i n the same animal have also been shown (Behrisch & Percy, 1974). The theory which these and other experiments have suggested i s that, i n evolutionary terms, stable environments induce l i t t l e enzymatic heterogeneity, whereas eurythermic conditions do, and this enables greater physiological f l e x i b i l i t y . Gene duplication, i n this way, provides a means for conservative modification of the basic model for protein function. What might be some of the properties contributing to greater functional capacity? Comparisons of pig heart and rainbow trout l i v e r 48 soluble NADP-linked i s o c i t r a t e dehydrogenases have shown that stable characteristics for the enzyme are molecular weights, cationic and cofactor requirements, i n h i b i t o r s p e c i f i c i t y , and activation energy. Properties which accounted for enzymic differences were electrophoretic mobility, and Km parameters with respect to temperature, cosubstrate, and a s p e c i f i c enhibitor, ADP (Moon, 1972). In G i l l i c h t h y e s , tissue s p e c i f i c enzymic-substrate-inhibitions of pyruvate kinases and lactate dehydrogenases indicated one means for selective channeling towards glycolysis (under cold, high 0^ conditions), or towards lactate formation (under high temperatures, low 0^ conditions) (Somero, 1973). S p e c i f i c i t y of enzymic action necessitates ordered chemical processes, which have been described by the concept of a one to one enzyme substrate interaction. However, the tissue s p e c i f i c differences and c a t a l y t i c f l e x i b i l i t y might suggest a more f l e x i b l e s t r u c t u r a l model for eurythermic enzymes. Increased structural f l e x i b i l i t y might have been derived from modifications of minor molecular groups i n the proteins which are s i g n i f i c a n t i n protein s t r u c t u r a l s t a b i l i t i e s . Changes i n weak bond interactions of the proteins would then render the enzymes more sensitive to modulators. Negative and positive cooperative effects by modulators have been found to be very s i g n i f i c a n t influences of enzymic a c t i v i t i e s (Behrisch § Johnson, 1974a; Moon, 1972). The consequence of these would be to extend the capacity for metabolic integration over wider ranges of change. This seems to be a reasonable tentative explanation for eurythermic adaptability that f i t s i n with current theories for enzyme function. Thus, instead of changes i n the complexity of enzymic forms or i n protein quantities, capacity for adaptation i s extended by increased s e n s i t i v i t y to a complexity of biochemical modulatory forces. 49 APPENDIX A 1. Polyacrylamide gel electrophoresis The process of polymerization of acrylamide and cross-link bisacrylamide i s i n i t i a t e d by TEMED. and ammonium persulfate or r i b o f l a v i n in the presence of l i g h t (Chrambach, 1971): CH2-CH C=0 I NH„ CH =CH 2 I C=0 NH NH2 NH I C=0 CH2=CH i n i t i a t o r s CH.-CH— CH -CH— CH--CH" I I 2 I I I Acrylamide bisacrylamide C=0 NH„ NH„ io C=0 ! NH„ NH„ I-2 C=0 CH2-CH" CH2-CH-C=0 I NH CH2 NH I C=0 CH2-CH polyacrylamide Pore, size (Davies, 1964) of the formed gel i s a function of both monomer and co-monomer concentrations. As the concentration of monomere i s increased, the concentration of co-monomere should be decreased i n order to maintain gel f l e x i b i l i t y . The equal increase or reduction of both components to extremes give gels that are too b r i t t l e or too soft. In low concentration gels, sucrose (40%) i s included to provide osmotic strength, as i s the case for stacking gels given i n the methods. Ribo-f l a v i n catalyst produces larger pore size than does ammonium persulfate, but these have a tendency to shrink with time. Stacking gels should therefore be made shortly before use, whereas separation gels can be stored overnight i f necessary. A 7.5% gel w i l l have an average pore size 50 o o of 50 A and a hydrated chain diameter of about 10 A. This provides some f r i c t i o n a l resistance for most c e l l u l a r proteins. Proteins between 200 o o A to 400 A are sharply separated, while larger and globular proteins are excluded (Ornstein, 1964). Most proteins exist i n t h e i r charged forms at the pH selected. Migration of proteins i s based upon ionic charge mobility. E l e c t r i c a l mobility i s d i r e c t l y proportional to the net charge and inversely proportional to the size of the molecule and the solution v i s c o s i t y ( i . e . f r i c t i o n a l resistance provided by pore size) (Ornstein, 1964). A system of ions and pH gradients was developed to f a c i l i t a t e the phenomenon of stacking and to f a c i l i t a t e e f f i c i e n t protein migration i n the running gel. I t has been argued by Richard and Lecanidou (1974) that the stacking phenomenon depends upon sample mass and buffer i o n i c strength alone and that stacking gel i s unnecessary to bring about reduction of sample volume into a th i n band at the running gel i n t e r -phase i n the i r studies of RNA, and discussion for protein. According to these authors the optimum load depends upon the i o n i c strength of buffers used and on the mobility of the anion, with increased loads possible at higher buffer concentrations. Nevertheless, stacking gels have been i n wide use and the phenomenon w i l l be considered i n greater d e t a i l i n order to understand the summary of necessary conditions for polyacrylamide gel electrophoresis given further down. According to Williams and Reisfeld (1964), the process of protein migration and resolution into bands during acrylamide gel electrophoresis i n a discontinuous buffer system i s explained as follows: By selecting a leading ion with highest mobility and a t r a i l i n g ion with mobility lesser than any protein, and a buffer pH discontinuity at the stacking running gel interphase, a voltage pH gradient i s set up 51 during electrophoresis which produces protein stacking. When current i s turned on, the leading ions i n the buffer migrate ahead, creating an increased voltage gradient i n respect to the slower migration species. This increased voltage gradient accelerates the slower migration species and a steady state migration band i s established whereby proteins become stacked i n a millimicron thick zone. When proteins enter the separation gel, migration i s influenced by sieving effects and the higher pH gradient. The leading and t r a i l i n g ions w i l l therefore migrate ahead of the proteins. The proteins w i l l become separated based upon t h e i r size and charge. The summary of the necessary conditions for anionic and cationic (in bracket) systems below i s from Williams and Reisfeld (1964): 1) pH of running gel should be such that proteins are bi o l o g i c a l l y stable and have the same sign of charge at that pH. 2) Chloride (potassium) ion i s used as leading ion. 3) T r a i l i n g ion i s a weak acid (weak base) or amino acid with a pK less than (greater than) the pH unit of the running cl gel. 4) pH of sample and stacking gel i s 2 to 3 pH units less than (greater than) the pK of the t r a i l i n g ion so that the t r a i l i n g ion i s weakly charged and therefore migrates slowly. 5) Buffer systems should have a pK a of equal or less (equal or greater) than one pH unit from the running gel to provide for good buffering capacity. 6) IN HC1 (KOH) i s used i n stock solution for making gels. It has been found by Poulik (1966) that the quality of protein separation i n acrylamide gel electrophoresis depends upon ionic concentrations for any system of ions used. Low i o n i c i t y of the buffer medium allowed for high potential gradients without adverse joule heating effects. According to Poulik better resolution w i l l also r e s u l t with low i o n i c strength since ion binding to proteins w i l l be reduced and i n t r i n s i c differences i n protein charges at a given pH w i l l not be masked. 2. Sodium dodecyl sulfate acrylamide gel electrophoresis For SDS gel ectrophoresis, proteins are f i r s t reduced i n mercaptoethanol and bond to sodium dodecyl sulfate (C^OSO^). This causes extensive disruption of hydrogen bonds, and hydrophobic and d i s u l f i d e linkages, which results i n s o l u b i l i z a t i o n of many r e l a t i v e l y insoluble proteins. Identical amounts of SDS are bound to the protein on a gram to gram basis by predominately hydrophobic binding (Shapiro et a l . , 1967; Fish ejt al_., 1970; Reynolds § Tanford, 1970). Reduction of d i s u l f i d e bonds i s required to optimize a v a i l a b i l i t y of hydrophobic residues and to obtain maximal binding. In the process, the native structure of the protein i s changed to an extended rod-like conformation with a moderately high content of c<-helex i n which hydrophobic residues are externally exposed for SDS binding (Nozaki ejt al_., 1974). The forces of attraction between the denatured protein and detergent are greater than between SDS, thus preventing the formation of detergent micelles (Tanford, 1968). Binding equilibrium i s established at less than 0.1% SDS concentrations (Nozaki et a l . , i b i d ) . The resultant SDS-protein complex i s highly ordered i n structure and has been found to have i d e n t i c a l hydrodynamic shapes d i f f e r i n g only i n dimensions (Stokes radius), which are dependent upon the protein molecular weight (Fish et a l . , i b i d ) . The SDS treated proteins are therefore separated i n electro-phoresis based upon thei r size differences alone. Plots of log Rg (Stokes radius) versus log molecular weights were found to be li n e a r for proteins i n the ranges 15,000-75,000 m wt (Fish et a l . , i b i d ) . At the lower molecular weight l i m i t s , protein-SDS complexes assume approximately spherical shapes instead of the rod l i k e p a r t i c l e s . The s l i g h t v a r i a b i l i t i e s observed i n the upper portions of the SDS gels ('a' i n the graphs) may be due to these lower l i m i t s of resolution, or other factors influencing separation at the region near the o r i g i n as discussed by Richter-Lansberg e_t al_. (1974). Factors discussed include reconstitution of d i s u l f i d e bridges by dissociation of dodecyl sulfate-protein complexes through sieving i n t h e i r system of discontinuous gel electrophoresis and protein mobility changes due to a l t e r a t i o n i n io n i c environment of the sample during electrophoresis. According to these authors some of these problems can be overcome by the addition of mercaptoethanol and sodium dodecyl sulfate to the upper buffer chamber to prevent reoxidation and dissociation. This was done i n the present studies. 54 APPENDIX B Densitometry graphs from polyacrylamide gel electrophoresis shown i n the results given i n section IV (pages 27-39), for G i l l i c h t h y e s  m i r a b i l i s . Numbers on graphs refer to gross peak areas and not to s p e c i f i c numbers of protein bands nor do they indicate unique separated units of protein. For a band to be labled as a unit protein, correlative studies of known proteins electrophoresed at the same time are necessary. Likewise, only r e l a t i v e quantitative comparisons should be made of peak areas,, keeping i n mind that due to density differences, minor bands w i l l appear as 'humps' off major peaks, or they can be obscured. b= bromophenol blue tracking dye. // = end of gel. 55 Graph 1. Gillichythes mirablis.Muscle 13 days acclimation. From f i g . 2, page 27. 56 Graph I I . Liver, non-frozen tissue. 13 days acclimation. Compare with graph I I I , quick frozen one week stored tissue from the same protein sample. From f i g . 4 a, page 29. 1 57 Graph III Liver, one week frozen stored tissue. From f i g . 4 b, page 29. 58 Graph IV. 28°C starved stressed - liver. From fig. 6, page 31. Graph V. 28 C starved stressed - muscle. From f i g . 6, page 31. 60 Graph VI. 28 C starved stressed - brain. From f i g . 6, page 31. 0Y O 61 Fig. V I I . 13 days acclimation From the same tissue as i n graph I I , but stored frozen over night i n homogenate form. Graphs of gels from f i g . 7 a, page 32. 62 Graph VIII. 20 days acclimation „..$From fig. 7 b, page 32, 1 63 Graph IX. 31 days acclimation From f i g . 8 a, page 33. 64 Graph X. 38 days acclimation From f i g . 8 b, page 33. ;- i i : . i SB Graph XI.. 45 days acclimation From f i g . 9, page 34. Duplicate gels for each set of temperatures. 6 6 i . • • Graph XII. Sodium dodecyl sulfate acrylamide gels ' j From f i g . 13 a, page 38.' 'a' = region i n the gels where minor bands are v i s i b l e . In the other numbered peaks areas the minor bands are less obvious-in these densitometry i ;.v*igraphs due to the great difference i n the density of protein stains. These minor bands are more obvious i n the. ' i • •/photograph. -> 67 BIBLIOGRAPHY Bailey, G. S. , Cocks, C. T. £ Wilson, A. C. 1969. 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