EFFECTS OF FREEZING ON PARTICULATE ENZYMES OF RAT LIVER*

Transcription

1 EFFECTS OF FREEZING ON PARTICULATE ENZYMES OF RAT LIVER* BY VIVIAN S. PORTER, NANCY P. DEMING, RITA C. WRIGHT, AND E. M. SCOTT (From the Arctic Health Research Center, United States Public Health Service, Anchorage, Alaska) (Received for publication, June 15, 1953) The fact that freezing caused marked changes in oxidative activity of tissues was of comparatively little interest until it became apparent that most if not all the enzymatic activities of cells could be reproduced under suitable conditions in cell-free systems. Isolated soluble enzymes were usually unaffect,ed or even stabilized by freezing, and damaging effects of freezing on other enzymes could be attributed to destruction of cells. Thus Lynen (I), in a study of frozen yeast cells, ascribed the effects of freezing to dilution of the enzymes and cofactors resulting from breaking of cell membranes. He also showed that diphosphopyridine nucleotide (DPN) restored activity of alcohol dehydrogenase after freezing. In a comparable study on animal tissues (a), he also ascribed the effects of freezing to breaking of cells. De Robertis and Nowinski (3, 4) showed that respiration and succinate oxidation not only of frozen liver slices, but also of frozen liver homogenates, were greatly reduced. Lynen (5), using kidney homogenates, obtained the same results and concluded that there were effects of freezing other than destruction of cell membranes. The sensitivity of particulate or mitochondrial enzyme systems to freezing has been a common observation. Loomis (6) reported a stabilization of such activity by fast freezing in the presence of substrate and sucrose. Huennekens and Green (7) showed that added DPN would restore some of the activity of frozen washed homogenates. Repeated slow freezing and thawing have been used in methods of preparation of pyruvic and a-ketoglutaric dehydrogenase (8, 9). Williams (lo), after freezing mitochondrial preparations, found an increased requirement for adenosinetriphosphate (ATP) in choline oxidation. The particulate oxidative enzymes of rat liver are largely inactivated by freezing. A detailed study of these enzymes was therefore undertaken, with the results described in this report. * Presented at the 122nd annual meeting of the American Chemical Society, Atlantic Cit,y, New <Jersey, Septenlher 11-19,

2 884 EFFECTS OF FREEZING ON ENZYMES Methods Enzyme Systems-Washed homogenates or mitochondria of rat liver were prepared by the methods of Green et al. (ll), Kennedy and Lehninger (12), and Hogeboom et al. (13), or by slight modifications of these procedures. Since the effects of freezing were found not to depend on method of preparation of the homogenate, most of these experiments were performed on washed homogenates prepared by a rapid method with magnesium chloride as a precipitant. Rat liver was homogenized in 0.04 M magnesium chloride and centrifuged for 5 minutes at 2200 X 9. The precipitate was twice washed by resuspending in 0.04 M magnesium chloride and centrifuging at 2200 X g for 3 minutes. An alternative method was to homogenize in 0.25 M sucrose and centrifuge for 10 minutes at 600 X g. The supernatant fluid was decanted, made 0.04 M with magnesium chloride, and centrifuged at 2200 X g for 10 minutes. The precipitate was then washed twice with 0.04 M magnesium chloride as above. Washed homogenates prepared with magnesium chloride showed enzymatic activities comparable to preparations from other procedures, while the yield of activity was higher. Freezing-The enzyme suspension was frozen by immersion of a flask in a dry ice-acetone bath until the suspension acquired a characteristic white, dry appearance. Oxygen Uptake-Each Warburg flask contained potassium phosphate buffer (ph 7.35) M, magnesium chloride 3.3 X 10e3 M, substrate M, DPN 1.0 X 1O-4 M, ATP M, nicotinamide 0.01 M, cytochrome c 3 X lop6 M, and homogenate 0.2 to 0.6 mg. of N per ml. In addition, when the substrate was pyruvate, octanoate, lactate, or propionate, the flask contained 0.01 M potassium bicarbonate; when malate, oxalacetate, aspartate, or octanoate was the substrate, each flask contained M magnesium chloride. Octanoate was used as substrate at a concentration of 6.7 X lo- M, and when succinate was studied the flask contained buffer M, cytochrome c 3 X lo M, succinate M, and homogenate 0.1 to 0.3 mg. of N per ml. The bath temperature was 30, and substrate and enzyme were both placed in the main compartment before equilibration. Although values for the blank oxygen uptake were usually insignificant, they were subtracted from uptakes obtained in the presence of substrates. RESULTS AND DISCUSSION The results of freezing on enzymatic activity of rat liver homogenate are shown in Table I. Each figure represents an average of three or more experiments. In no case was enzyme activity unaffected by freezing, and the presence or absence of added coenzymes had an important influence on the result.

3 PORTER, DEMING, WRIGHT, AND SCOTT 885 Ej ects of Coenzymes-Except for the oxidation of succinate, in every case in which sufficient activity could be recovered to test the effect of DPN, an increased requirement for DPN was found after freezing. Data on oxidation of L-malate after freezing have been previously reported (7). In agreement with this report, endogenous DPN was no longer precipitable TABLE Effect of Freezing on Oxidative Activity of Washed Rat Liver Homogenate Oxygen uptake of frozen homogenate as per cent of oxygen uptake of unfrozen homogenate with addition of the same coenzymes. The figures in parentheses represent oxygen uptake of unfrozen homogenate with the coenzyme lacking as per cent of oxygen uptake of unfrozen homogenate with all three coenzymes present. Recovery of activity after freezing Unfrozen ; Substrate complete I- system* Complete DPN ATP Cytochrome systemt omitted omitted c omitted Succinate$... Citrate.... (I g.... Acetaldehyde... L-Proline.... L-Glutamate.... Choline chloride.... p-hydroxybutyrate... L-Malate.... on-lactate... a-ketoglutarate.... Oxalacetate... L-Aspartate.... Propionate.... Pyruvate... Octanoate I 13 (53) 25 (41) 16 (98) 14 (63) 115 (87) 22 (98) 9 (109) 6 (59) 7 (57) 5 (88) 0 (100) 4 (62) * Microliters of oxygen per hour per mg. of nitrogen. t Containing added ATP, DPN, and cytochrome c. $ ATP and DPN were not added in succinate oxidation. $ TPN substituted for DPN. 18 (25) 22 (19) 61 (84) 51 (74) 50 (39) 66 (198) 74 (125) 25 (55) 27 (100) (6) 17(4F 91 (73) 86 (66) 56 (43) 49 (39) 112 (103) 85 (100) 32 (38) 25 (101) 25 (68) by centrifuging after freezing, although the oxidase activity itself was. Freezing had no effect on DPN content itself nor on DPN nucleosidase. Since the DPN content was unchanged by freezing, the DPN present must have become less effective. Ochoa ((14) p. 60) has cited other cases in which coenzymes after dissociation are required in larger concentrations than were originally present in the holoenzyme. There was no indication of an increased requirement for ATP after freezing except in the cases of the substrates citrate and choline. A detailed

4 886 EFFECTS OF FREEZING ON ENZYMES study of the latter has already been reported by Williams (lo), and, since our results are in agreement, they will not be discussed here. A study was made of the effects of substitution of adenosinediphosphate (ADP) and adenosinemonophosphate (AMP) for ATP. The averages of three experiments are shown in Table II. ADP gave almost the same results as ATP after freezing. On the other hand, AMP was wholly ineffective after freezing. Although AMP had an activating effect almost equal to or somewhat greater than that of ATP before freezing, after freezing the result was the same as if no adenylic compound were present at all. The suggestion has been made that AMP, in common with other phosphate TABLE Comparative Efects of ATP, ADP, and AMP on Oxidative Activity after Freezing Oxygen uptake of frozen homogenate as per cent of oxygen uptake of unfrozen homogenate. The figures in parentheses represent oxygen uptake of unfrozen homogenate as per cent of oxygen uptake when ATP was present. Citrate. Acetaldehyde. L-Proline L-Glutamate. Choline chloride. t. fl-hydroxybutyrate II I Recovery of activity after freezing Complete system* * Containing cytochrome c, DPN, and ATP. t Data of Williams (10). AMP instead of ATP 65 (91) 16 (SO) 74 (117) 49 (109) 37 (107) 39 (89) 31 (108) 31 (127) 147 (101) 60 (174) 106 (114) 130 (120) 89 (120) 70 (125) No ATP 18 (30) 71 (72) 47 (68) 46 (51) 58 (235) 78 (151) 72 (116) acceptors, activates by permitting more oxidative phosphorylation (15). and, since phosphorylation has never been demonstrated by us after freezing, the present results are in accord with this suggestion. An alternative possibility is that AMP is not an activator until it has been phosphorylated to ADP or ATP. ATPase of washed liver homogenate was unaffected by freezing.l It was thought that the increased requirement for ATP and DPN after freezing, as shown in oxidation of citrate, might be due to an increased requirement for triphosphopyridine nucleotide (TPN), since washed ho- 1 Inorganic phosphate of unknown origin was apparently formed by the freezing process. Trichloroacetic acid filtrates of unfrozen mitochondrial preparations contained about 10 y of P per mg. of N. After freezing, this value was increased by 50 per cent.

5 PORTER, DEMING, WRIGHT, AND SCOTT 887 mogenates of this type have been shown to catalyze the transformation of DPN and TPN in the presence of ATP (16). However, when TPN was substituted for DPN, there was still an increased requirement for ATP in citrate oxidation after freezing (Table I). ATP must then have some specific function in citrate oxidation other than the transformation of DPN to TPN. There was no increased requirement for cytochrome c after freezing except in the case of oxidation of succinate. Since this oxidation was 2 to 10 times as rapid as any of the others studied, it might be expected that this coenzyme would not be limiting for other oxidations. The situation was not this simple, however, because the oxidation of other substrates by unfrozen homogenates was considerably lower when cytochrome c was not added. In the oxidation of citrate, acetaldehyde, and proline, the reaction rate was limited more by omission of cytochrome c before freezing than afterward. This resulted in a higher per cent recovery without added cytochrome c than with it, although the absolute recovery was about the same. After prolonged centrifuging, absorption bands corresponding to reduced cytochrome c were found in the supernatant solution of frozen washed homogenate.2 It would therefore appear that except in the case of succinic dehydrogenase, endogenous cytochrome c is no more effective than added cytochrome c. No other coenzymes had any effect whatever on recovery of oxidative activity of washed homogenate of rat liver after freezing. Those tested included coenzyme A, flavin-adenine dinucleotide, cocarboxylase, pyridoxal phosphate, and boiled enzyme extracts. Freezing Whole Liver-When liver was frozen immediately after removal from the animal and a washed homogenate then prepared, the oxidative activity of the homogenate was qualitatively similar to that found when the homogenate was prepared first and then frozen, although the yields of activity were low. Efect of Sucrose-Treatment with glycerol has been found to preserve the viability of spermatozoa during freezing (17). A study of the effects of glycerol, ethylene and propylene glycols, and sucrose in 30 per cent concentration on preservation of activity of washed liver homogenates was made. All of these compounds acted to prevent the destructive action of freezing. Ethanol (30 per cent), dioxane (30 per cent), potassium chloride (25 per cent), and serum albumin (15 per cent) were ineffective. Treatment of the homogenate with high concentrations of glycerol or gly- * A considerable quantity of material capable of reducing cytochrome c was found in these systems immediately after freezing. Cytochrome c added in more than 50 times the amount found in the supernatant solution was reduced by frozen homogenate.

6 888 EFFECTS OF FREEZING ON ENZYMES cols inactivated certain of the enzymes, but sucrose did not have this effect. Accordingly, only the effect of sucrose was studied in detail. In Table III are shown the results of single experiments in which the homogenate was frozen in 30 per cent sucrose. Even in the absence of DPN, good recoveries of activity were obtained. The protective action of sucrose was evident at relatively low concentrations, becoming evident at 4 or 5 per cent and maximal at 10 per cent. It was therefore possible to test the effect of freezing in sucrose on oxidative phosphorylation. After TABLE Effect of Presence of 30 Per Cent Sucrose on Freezing Washed Liver Homogenate Oxygen uptake of frozen homogenate as per cent of oxygen uptake of unfrozen homogenate. The figures in parentheses are oxygen uptake of unfrozen homogenate treated with 30 per cent sucrose as per cent of oxygen uptake of untreated unfrozen homogenate. Succinate Citrate. Acetaldehyde.. L-Proline L-Glutamate. Choline chloride. n-malate nn-lactate a-ketoglutarate Pyruvate Octanoate. Substrate III Added Cytochrome - c coenzymes ATP Sucrose absent* jucrose present 71 (98) 100 (87) 71 (63) 91 (110) 90 (70) 86 (110) 93 (81) 86 (101) 98 (41) 98 (49) 87 (62) * Sucrose absent during freezing. Final sucrose concentration in all Warburg flasks, 2 per cent. treatment with 30 per cent sucrose for a brief period, no oxidative phosphorylation could be demonstrated in washed liver homogenates, whether frozen or not. After the homogenate was frozen in 8.5 per cent sucrose, 65 per cent recovery of oxidative phosphorylation with a-ketoglutarate as substrate was obtained. Recovery of oxygen uptake was somewhat higher, and the P:O ratio was thus lowered by freezing. A microscopic examination was made of washed homogenate frozen by dry ice, in the presence and absence of sucrose. In the absence of sucrose, crystallization began at relatively few nuclei, and large elongated crystals were formed. After thawing, the enzyme-bearing particles were no longer discrete units, but had been compressed into strands between the crystals. With sucrose, crystallization began at many nuclei, and the crystals were

7 PORTER, DEMING, WRIGHT, AND SCOTT 889 but little larger than the enzyme-bearing particles. After thawing, these particles remained apparently unchanged. E$ect of Substrate-When the washed homogenate was frozen in the presence of 0.25 M substrate, there was an increased recovery of activity. Table IV shows the results of single experiments. The effect of substrate was found whether DPN was present or not. A substrate concentration of 0.01 M had a less definite protective action. Freezing in the presence of one substrate protected the capacity to oxidize not only that substrate, but other substrates as well. The protective action of substrate could not TABLE Eflect of Presence of 0.25 M Substrate on Freezing Washed Liver Homogenate Oxvaen untake of frozen homogenate as uer cent of that of unfrozen homogenate. Cytoc hiomec and ATP were added to all flasks except as noted. Coenzyme added... Substrate presentt... Succinate... Citrate... Acetaldehyde... L-Proline... L-Glutamate... Choline chloride.... L-Malate... DL-Lactate.... a-ketoglutarate.... Oxalacetate... Pyruvate... - IV i * DPN was added or omitted in the Warburg flasks except with choline and succinate. In the former case, ATP was added or omitted; in the latter, cytochrome c. t During freezing. Final substrate concentration in all flasks, M. be duplicated by removal of oxygen by evacuation of the enzyme before freezing. Speed and Extent of Freezing-When a washed homogenate was first frozen at -10 (1 hour) and then in dry ice-acetone, the extent of damage was less than when the homogenate was frozen directly in dry ice-acetone. Still less destruction was found when the enzyme was frozen only at The latter temperature was above the eutectic temperature of the salts present. Freezing was visibly not complete, and the ice mass had a moist appearance. Conclusions There is no evidence that the effect of freezing here observed is other than a mechanical one, in which enzyme-bearing particles are modified by

8 890 EFFECTS OF FREEZING ON ENZYMES compression and shearing between ice crystals. Slow freezing would be less damaging because the stresses produced would be less, even though crystal size was larger. Freezing in sucrose would be less damaging because of smaller crystal size. Sucrose could hardly have much dehydrating effect, since it is active at isotonic or hypotonic concentrations. The reason for the protective action of substrate does not appear to have any simple interpretation. The known ways in which the particles are modified by freezing consist of a loss of identity of the particles, release of coenzymes, and rendering certain proteins soluble. An obvious hypothesis to explain the effects of coenzymes is an extension of Lynen s idea from cells to cell particles. If the enzyme-bearing particles have a membrane which is destroyed by freezing (or for that matter, any organized structure which is disrupted by freezing), the enzymes and coenzymes would be released and a dilution effect obtained. In any consecutive reaction sequence, the rate of the over-all reaction would be higher if all enzymes and coenzymes were localized in small units, rather than distributed throughout the solution. This dilution hypothesis, while applicable to several of the oxidations studied, does not explain why some of the reactions are apparently irreversibly lost by freezing, unless it is assumed that new, and as yet unknown, cofactors are involved. The one coupled reaction measured, oxidative phosphorylation, was stopped by freezing, and it is possible that some of the oxidations studied are coupled reactions, which are also completely prevented by freezing. There is a lack of independent evidence for this point of view, however. SUMMARY 1. Freezing with dry ice caused marked reductions in the capacities of washed rat liver homogenates to oxidize most substrates. Under optimal conditions, only seven out of fifteen oxidative activities of rat liver showed recoveries of better than 50 per cent after freezing. Oxidative phosghorylation stopped completely after freezing. 2. Almost all oxidative activities showed some recovery after freezing on addition of pyridine nucleotides. Furthermore, added cytochrome c aided recovery of succinate oxidation, and added ATP aided recovery of choline and citrate oxidation. Other coenzymes had no effect. 3. Freezing in sucrose solution prevented most of the destructive effects. Freezing in the presence of substrate was less effective. 4. Slow freezing was less destructive than fast freezing. 5. It is suggested that the effect of freezing may be a mechanical one, in which essential elements of structure in enzyme-bearing particles are disrupted by physical forces of shear between ice crystals.

9 PORTER, DEMING, WRIGHT, AND SCOTT 891 BIBLIOGRAPW 1. Lynen, F., Ann. Chem., 639, 1 (1939). 2. Lynen, F., 2. physiol. C&m., 264, 146 (1940). 3. de Robertis, E., and Nowinski, W. W., Rev. Sot. urgent. biol., 18, 333 (1942). 4. Nowinski, W. W., and de Robertis, E., Rev. Sot. urgent. biol., 19, 527 (1943). 5. Lynen, F., 2. physiol. Chem., 281, 83 (1944). 6. Loomis, W. F., Arch. Biochem., 26, 355 (1950). 7. Huennekens, F. M., and Green, D. E., Arch. Biochem., 27, 418 (1950). 8. Jagannathan, V., and Schweet, R. S., J. Biol. Chem., 196, 551 (1952). 9. Sanadi, D. R., Littlefield, J. W., and Bock, R. M., J. BioZ. Chem., 197,851 (1952). 10. Williams, J. N., Jr., J. Biol. Chem., 198, 579 (1952). 11. Green, D. E., Loomis, W. F., and Auerbach, V. H., J. Biol. Chem., 172,389 (1948). 12. Kennedy, E. P., and Lehninger, A. L., J. Biol. Chem., 179,957 (1949). 13. Hogeboom, G. H., Schneider, W. C., and Pallade, G. E., J. BioZ. Chem., 172, 619 (1948). 14. Ochoa, S., PhysioE. Rev., 31, 56 (1951). 15. Lardy, H. A., and Wellman, H., J. BioZ. Chem., 196, 215 (1952). 16. Katchman, B., Betheil, J. J., Schepartz, A. I., and Sanadi, D. R., Arch. Biothem. and Biophys., 34, 437 (1951). 17. Polge, G., Smith, A. V., and Parkes, A. S., Nature, 164,666 (1949).

10 EFFECTS OF FREEZING ON PARTICULATE ENZYMES OF RAT LIVER Vivian S. Porter, Nancy P. Deming, Rita C. Wright and E. M. Scott J. Biol. Chem. 1953, 205: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1