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Urea-mediated freeze-thaw hybridization of lactate dehydrogenase
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
45
35 I I I
UREA-MEDIATED F R E E Z E - T H A W HYBRIDIZATION OF LACTATE D E H Y D R O G E N A S E EDWARD j . MASSARO
Department of Biology, Yale University, New Haven, Conn. (U.S.A.) (Received April 6th, 1967)
SUMMARY
The subunits of lactate dehydrogenases from a wide variety of related and unrelated organisms can be reassociated in vitro into functional "hybrid molecules". This can be accomplished by subjecting isozyme mixtures to a freeze-thaw cycle in phosphate buffer containing the necessary hybridization-promoting substances: I-, Br-, CI-, thiocyanate, or i-anilinonaphthalene-8-sulfonate. It has been observed now that low concentrations of urea (less than o.I M) in sodium phosphate buffer also will promote freeze-thaw hybridization. Urea and the other hybridizationpromoting substances appear to function by a similar mechanism. It has been suggested that urea disrupts protein structure by an ion-exchange mechanism. It has also been observed that Tris not only inhibits freeze-thaw hybridization, but also protects the enzyme against freeze-thaw destruction. Under various conditions, the isozymes possessing the least net negative charge are more susceptible to irreversible freeze-thaw denaturation than the other isozymic types. This may be the result of evolutionarily preserved structural characteristics common to all isozymic forms.
INTRODUCTION
Reassociation in vitro of the subunits of lactate dehydrogenases from a wide variety of related and unrelated organisms produces functional hybrid moleculess,°. Several methods have been described for attaining molecular hybridization1-4,6,8. These may be classified into two categories: (a) reversible enzyme denaturation by urea, guanidine-HC1, or LiC1 at temperatures above the eutectic point and (b) freezethaw-induced hybridization. The mechanism by which each category of methods functions would appear to be unrelated. However, the demonstration that low concentrations of urea will promote freeze-thaw hybridization suggests that the two categories may be fundamentally similar. Evidence in support of this hypothesis will be presented in this report. Abbreviation: LDH, lactate dehydrogenase; the isozymes possessing the greatest net negative charge are designated LDH-I and the isozymes possessing the least net negative charge are designated LDH- 5.
Biochim. Biophys. Acta, 147 (1967) 45-51
46
E.J. MASSA~O
MATERIALS AND METHODS
Enzyme sources. Lactate dehydrogenase was obtained from tissues of the horse (Equus caballus), flying fish (Exonautes rondeletii), and shark (Carcharrinusplatyodon). Enzyme preparation. Selected tissues from each organism were minced and homogenized in appropriate quantities of glass-distilled water or neutral o.i M TrisHC1. The homogenate was cleared by centrifugation at 16300 × g for 20 rain at 4 ° and the fat layer was removed by filtering the supernatant through a fast grade of filter paper. The enzyme was recovered from the filtrate by ammonium sulfate fractionation. Highly purified individual isozymes of lactate dehydrogenase were isolated from suctl crude enzyme preparations by repeated column chromatography on DEAE-cellulose or by extraction from starch gel following electrophoresis. These isozyrnes migrated as a single band both in starch and acrylamide gel and in the ultracentrifuge. However, it is well established that molecular hybridization can be accomplished readily in crude enzyme preparations. The results of such experiments, measured by any criterion, are comparable to those attained utilizing highly purified, individual isozymes. Therefore, partially purified isozymes were used in many of the experiments reported here. Electrophoresis. The starch gel electrophoretic technique and the tetrazolium method for detecting lactate dehydrogenase activity have been described previously TM. Assays. Enzymatic activity and protein concentration were measured as previously reported 1°. Reagents. All chemicals used in these experiments were of the highest quality commercially available and, with the exception of urea which was recrystallized twice from methanol, were not further purified. RESULTS
Figs. I and 2, respectively, are examples of intraspecific (between shark isozymes) and interspecific (between flying fish and horse isozymes) molecular hybridization. In both of these cases, subunit reassociation was achieved by freezing and thawing the isozyme mixtures in neutral sodium phosphate buffer containing either C1- or urea3,8,1°. This demonstrates that CI-, and the other freeze-thaw hybridizationpromoting substances (Br-, I-, thiocyanate, I-anilinonaphthalene-8-sulfonate)* can be replaced by relatively low concentrations (less than 0.I M) of urea. The characteristics of urea mediated freeze-thaw hybridization are similar to those of halide or thiocyanate-mediated hybridizationS,S,TM. Thus, urea promotes hybridization in the presence of phosphate ions, but, at the same concentrations, is without effect in the absence of phosphate (e.g. in distilled water). Also, Tris blocks hybridization even at urea concentrations many fold greater than required to induced hybridization in the presence of phosphate. Tris stabilizes the enzyme against freeze-thaw mediated destruction. The mechanism by which this is accomplished is unknown. It is not a function of ionic strength. Lactate dehydrogenase is considerably more resistant to freeze-thaw mediated urea " " P r o m o t e r " s u b s t a n c e will be defined o p e r a t i o n a l l y as a n y c h e m i c a l a g e n t w h i c h f u n c t i o n s in c o n j u n c t i o n w i t h o t h e r c h e m i c a l a g e n t s to induce f r e e z e - t h a w r e a s s o c i a t i o n of s u b u n i t s .
Biochim. Biophys. Acta, 1:47 (1967) 45-51
UREA-MEDIATED HYBRIDIZATIONOF L D H
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(+)
ORIGIN
(--) b
c
d
e
f
g
h
Fig. i. Freeze t h a w h y b r i d i z a t i o n of s h a r k l a c t a t e d e h y d r o g e n a s e i s o z y m e s in t h e pre s e nc e of NaC1 or urea. A l i q u o t s of e q u a l e n z y m a t i c a c t i v i t y of t h e m o s t n e g a t i v e l y c h a r g e d i s o z y m e ( L D H - i ) a n d t h e m o s t p o s i t i v e l y c h a r g e d i s o z y m e (LDH-5) were e m p l o y e d . The buffer s y s t e m w a s n e u t r a l o.I M s o d i u m p h o s p h a t e . F r e e z i n g t i m e was 16 h a t - - 2 o °. All s a m p l e s were t h a w e d a t r o o m t e m p e r a t u r e . H y b r i d i z a t i o n was d e t e c t e d b y s t a r c h - g e l e l e c t r o p h o r e s i s a t r o o m t e m p e r a t u r e for 6 h as p r e v i o u s l y d e s c r i b e d l ° . a = L D H - I ; b - LDH-5;c = LDH-I × LDH-5;d = c(LDH-I × LDH-5) + o . o i 5 MNaC1; e = c + o.oo7 M NaC1; f = c + o.oo3 M u r e a ; g = c + o . o o i 5 M u r e a ; h -- c + o.ooo 7 M urea. Due to t h e fact t h a t t h e i s o z y m e s e m p l o y e d in t h i s a n d m o s t of t h e subs e q u e n t e x p e r i m e n t s were c o n t a m i n a t e d w i t h n o n - l a c t a t e d e h y d r o g e n a s e p r o t e i n (see t e xt ), t h e r a t i o s of t h e c o n c e n t r a t i o n of NaC1 or u r e a to e n z y m e p r o t e i n c oul d n o t be c a l c u l a t e d . To p a r t i a l l y c o m p e n s a t e for t h i s deficiency, s e v e r a l d i l u t i o n s of t h e p r o m o t e r s u b s t a n c e s were e m p l o y e d so t h a t a r o u g h e s t i m a t e of t h e i r r e l a t i v e effectiveness could be o b t a i n e d . I t is to be n o t e d t h a t h y b r i d i z a t i o n of s h a r k L D H - I a n d L D H - 5 r e s u l t s in t h e f o r m a t i o n o n l y of 2 i s o z y m e s of i n t e r m e d i a t e m o b i l i t y where, on t h e basis of a s i m p l e b i n o m i a l r e c o m b i n a t i o n , 3 w o u l d be e x p e c t e d . Since i n t e r s p e c i f i c h y b r i d i z a t i o n s t u d i e s h a v e d e m o n s t r a t e d t h a t b o t h s h a r k L D H - I a n d L D H - 5 are h o m o p o l y m e r i c t e t r a m e r s , t h e s e r e s u l t s are difficult to e xpl a i n.
destruction in glass distilled water or Tris than in neutral phosphate buffer even at concentrations of phosphate too low to promote hybridization. Not all isozymic forms of lactate dehydrogenase undergo the same degree of freeze-thaw destruction. At concentrations of enzyme employed in these experiments (approx. o. 7 mg/ml), LDH-5 selectively is destroyed. Selective destruction is enhanced at low phosphate concentrations b y the other promoter substances. In a Tris-HC1 system, the urea concentration m a y be increased to relatively high levels, compared to a phosphate buffer system, with no visible effect on the stability of the enzyme. However, a critical urea concentration (differing for enzymes derived from different sources) exists beyond which LDH-5 is selectively destroyed. Increasing urea concentration above the level Biochim. Biophys. Acta, 147 (1967) 45 51
E. J. MASSARO
48
(+)
0
ORIGIN
(-) a
b
c
d
e
Fig. 2. I n t e r s p e c i f i c f r e e z e - t h a w h y b r i d i z a t i o n of l a c t a t e d e h y d r o g e n a s e i s o z y m e s in t h e p r e s e n c e of NaC1 or urea. A I i q u o t s of e q u a l e n z y m a t i c a c t i v i t y of horse L D H - I a n d fl yi ng fish s k e l e t a l m u s c l e L D H were e m p l o y e d . The buffer s y s t e m was n e u t r a l o . i M s o d i u m p h o s p h a t e . F r e e z i n g t i m e was 15 h a t 20 °. All s a m p l e s were t h a w e d a t room t e m p e r a t u r e . H y b r i d i z a t i o n w a s d e t e c t e d b y s t a r c h - g e l e l e c t r o p h o r e s i s a t r o o m t e m p e r a t u r e for 6 h as p r e v i o u s l y described 1°. a = hors e L D H - I ; b -- flying fish m u s c l e l a c t a t e d e h y d r o g e n a s e ; c -- horse L D H - I × flying fish m u s c l e L D H ; d = horse x fish + o . s M NaC1; e = horse × fish + o . o 6 M urea. The h y b r i d i s o z y m e s are i n d i c a t e d b y s m a l l circles.
effecting LDH-5 destruction results in progressive destruction of the other isozymic forms, utilimately including LDH-I. Preliminary evidence indicates that the order of lability of lactate dehydrogenase isozymes is L D H - 5 > L D H - 4 ~ L D H - 3 LDH-2 > LDH-I. The underlying reason for the increased susceptibility of LDH-5 to freeze-thaw destruction is unknown but appears to stem from structural characteristics common to LDH-5 from m a n y diverse species. Further evidence for a similar mode of action of urea and the halides in the hybridization mechanism has been obtained from complementation experiments. Biochim. Biophys. Acta, 147 (1967) 4 5 - 5 I
UREA-MEDIATED HYBRIDIZATION OF L D H
49
After establishing the minimal urea and NaCI concentration at which unequivocal hybridization could be elicited between shark L D H - I and LDH-5, identical isozyme mixtures were frozen and thawed in the presence of subthreshold concentrations both of urea and NaC1. It was observed that urea and NaC1 act in an additive manner and the hybridization which is obtained is indistinguishable from that produced by either promoter substance alone. Other isozyme combinations give similar results. Finally, F - does not function as a promoter substance 3 and subthreshold concentrations of urea in the presence of fluoride (up to I.O M) do not induce hybridization. As with the interactions of proteins in general, it is not the molar concentrations per se of the components of the hybridization-inducing environment which are of primary significance, but the molar ratios of these substances to enzyme protein. Since, in m a n y of the experiments described, isozyme preparations containing varying quantities (less than IO %) and types of non-lactate dehydrogenase proteins were utilized, no meaningful calculation of these ratios is possible. However, the relative effectiveness of urea in promoting freeze-thaw hybridization in impure enzyme preparation can be estimated. By comparing the minimal molar concentration of NaC1 necessary to induce hybridization in the phosphate system to that of urea, it has been calculated that urea is from two to ten times as effective as NaC1, depending upon the source of the isozymes. Preliminary evidence with highly purified mammalian isozymes indicates that these estimates are reasonably accurate. DISCUSSION
Recombination of lactate dehydrogenase subunits through reversible denaturation at supra-eutectic temperatures employs high concentrations of denaturant to produce dissociation of the tetramer2,4, e. Renaturation is accomplished b y dilution. An analogous cycle of concentration and dilution occurs during freeze-thaw hybridization. Due to the rapid rate of reassociation, the degree of structural perturbation involved in freeze-thaw hybridization must be small. This suggests that only small changes in conformation are necessary to achieve subunit dissociation at supraeutectic temperatures. However, the conditions for achieving these changes are such that high reagent concentrations must be employed. This results in protein denaturation whichis secondary to, although perhaps simultaneous with, subunit dissociation. Freeze-thaw hybridization is known to occur only under a limited number of conditions. The role of each of the components of the hybridization milieu is obscure. Diverse forces are responsible for quaternary structure and the nature of the promoter substances indicates that they act upon different types of forces. However, the same net result is produced. Apparently, selective disruption of certain of these forces will result in reversible dissociation. I t has been suggested that urea m a y function b y an ion-exchange mechanism to disrupt intramolecular bonds 5. In solution, urea m a y exist as a zwitterion with a large, positive, low density, head charge on the nitrogens and an intense negative charge on the oxygen. Under the proper conditions, the low density charge favors binding of the urea "cation" to the surface of macromolecules. This binding would be tenacious and the "cation" would function as a "primer" which dissociates or is displaced b y other functional groups (e.g. water). Halides and other promoter substances m a y function in a similar manner but at different sites. This is Biochim. Biophys. Acta, 147 (1967) 45-5I
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E. J. MASSARO
supported by the observation that the order of effectiveness of the halides in promoting freeze-thaw hybridization (I- > B r - > C1 ) is directly related to the charge density on the ion (I < B r - < C1-). Phosphate, arsenate, and nitrate also might function via a similar ion-exchange mechanism. A voluminous low density charge on the Tris ion, dimethyl sulfoxide, and certain polyhydroxy organic compounds could explain the mechanism by which they inhibit hybridization and protect the enzyme against freeze-thaw mediated urea denaturation. Tile binding of a large number of Tris ions on the surface of the macromolecule would form a "protective" coat against urea and the other promoter substances. A similar argument could be evoked to explain the greater effectiveness of sodium phosphate than potassium phosphate systems in promoting freeze-thaw hybridization a. Because of the larger low density charge on K ~, it might form a "protective" coat over the enzyme which would inhibit the penetration of phosphate and the other promoter substances. The greater susceptability of the LDH-5 type isozymes to freeze-thaw denaturation is difficult to explain since this observation is not limited to homologous isozynles of a single group of closely related organisms. On the contrary, similar results have been observed among the lactate dehydrogenase isozymes of a wide variety of organisms such as the cow, rabbit, horse and shark. Since it has been shown unequivocally that subunit recombination among lactate dehydrogenase isozymes derived from unrelated species is the rule rather than the exception 9, it m a y be that certain structural features of a given lactate dehydrogenase isozymes type are very similar in all species. It would be these features which impart to the molecule the properties characteristic of a particular type of lactate dehydrogenase isozymeL To nlaintain the identity of a given isozyme type, these features would have to be conserved throughout evolution and, in effect, would serve to "dictate" the "acceptable" amino acid substitutions within the basic structure of the molecule which obviously have occurred. The terms "dictate" and "acceptable" are used to emphasize the idea that, should the characteristic structural features of a given isozyme type be modified significantly, the isozyme type would cease to exist or the molecule might cease to function as a lactate dehydrogenase n. Thus, the concept of characteristic structural features implies that certain conformational aspects of a given lactate dehydrogenase isozyme type are universally similar. Such similarities easily account for the selective denaturation of homologous lactate dehydrogenase isozymes. ACKNOWLEDGEMENTS
This investigation was supported by N.S.F. research grant GB 544oX. The technical assistance of Miss S. MOORE is gratefully acknowledged. I am indebted to Professor C. L. MARKERT, Department of Biology, Yale University and to Dr. G. COLACICCO, Department of Biochemistry, Albert Einstein College of Medicine for helpful discussions during the course of this investigation. REFERENCES I S. ANDERSON AND (~. WEBER, Arch. Biochem. Biophys., 116 (1966) 207. 2 0 . P. CHILSON, L. A. COSTELLO AND N. O. I(APLAN, dr. ]!/[ol. Biol., IO (1964) 349. 3 0 . P. CHILSON, L. A. COSTELLO AND N. O. KAPLAN, Biochemistry, 4 (1965) 271.
Biochim. Biophys. Acta, 147 (1967) 45-51
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4 0 . P. CHILSON, G. B. KITTO, J. PUDLES AND N. O. KAPLAN, J. Biol. Chem., 241 (1966) 2431. 5 G. COLACICCO, Nature, 198 (1963) 583. 6 C. J. EPSTEIN, M. M. CARTER AND R. F. GOLDBERGER, Biochim. Biophys. Acta, 92 (1964) 391. 7 N. O. KAPLAN AND M. M. CIOTTI, Ann. N . Y . Acad. Sci., 94 (1961) 7Ol. 8 C. L. 1ViARKERT, Science, 14o (1963) 1329. 9 C. L. MARKERT, Harvey Lectures Ser., 59 (1965) 187. IO C. L. MARKERT AND E. J. MASSARO, Arch. Biochem. Biophys., 115 (1966) 417 . I1 K. E. NEET AND D. E. KOSI-ILAND, Jr., Proc. Natl. Acad. Sci. U.S., 56 (1966) I6o6.
Biochim. Biophys. Acta, 147 (1967) 45-51
BBA
45
35 I I I
UREA-MEDIATED F R E E Z E - T H A W HYBRIDIZATION OF LACTATE D E H Y D R O G E N A S E EDWARD j . MASSARO
Department of Biology, Yale University, New Haven, Conn. (U.S.A.) (Received April 6th, 1967)
SUMMARY
The subunits of lactate dehydrogenases from a wide variety of related and unrelated organisms can be reassociated in vitro into functional "hybrid molecules". This can be accomplished by subjecting isozyme mixtures to a freeze-thaw cycle in phosphate buffer containing the necessary hybridization-promoting substances: I-, Br-, CI-, thiocyanate, or i-anilinonaphthalene-8-sulfonate. It has been observed now that low concentrations of urea (less than o.I M) in sodium phosphate buffer also will promote freeze-thaw hybridization. Urea and the other hybridizationpromoting substances appear to function by a similar mechanism. It has been suggested that urea disrupts protein structure by an ion-exchange mechanism. It has also been observed that Tris not only inhibits freeze-thaw hybridization, but also protects the enzyme against freeze-thaw destruction. Under various conditions, the isozymes possessing the least net negative charge are more susceptible to irreversible freeze-thaw denaturation than the other isozymic types. This may be the result of evolutionarily preserved structural characteristics common to all isozymic forms.
INTRODUCTION
Reassociation in vitro of the subunits of lactate dehydrogenases from a wide variety of related and unrelated organisms produces functional hybrid moleculess,°. Several methods have been described for attaining molecular hybridization1-4,6,8. These may be classified into two categories: (a) reversible enzyme denaturation by urea, guanidine-HC1, or LiC1 at temperatures above the eutectic point and (b) freezethaw-induced hybridization. The mechanism by which each category of methods functions would appear to be unrelated. However, the demonstration that low concentrations of urea will promote freeze-thaw hybridization suggests that the two categories may be fundamentally similar. Evidence in support of this hypothesis will be presented in this report. Abbreviation: LDH, lactate dehydrogenase; the isozymes possessing the greatest net negative charge are designated LDH-I and the isozymes possessing the least net negative charge are designated LDH- 5.
Biochim. Biophys. Acta, 147 (1967) 45-51
46
E.J. MASSA~O
MATERIALS AND METHODS
Enzyme sources. Lactate dehydrogenase was obtained from tissues of the horse (Equus caballus), flying fish (Exonautes rondeletii), and shark (Carcharrinusplatyodon). Enzyme preparation. Selected tissues from each organism were minced and homogenized in appropriate quantities of glass-distilled water or neutral o.i M TrisHC1. The homogenate was cleared by centrifugation at 16300 × g for 20 rain at 4 ° and the fat layer was removed by filtering the supernatant through a fast grade of filter paper. The enzyme was recovered from the filtrate by ammonium sulfate fractionation. Highly purified individual isozymes of lactate dehydrogenase were isolated from suctl crude enzyme preparations by repeated column chromatography on DEAE-cellulose or by extraction from starch gel following electrophoresis. These isozyrnes migrated as a single band both in starch and acrylamide gel and in the ultracentrifuge. However, it is well established that molecular hybridization can be accomplished readily in crude enzyme preparations. The results of such experiments, measured by any criterion, are comparable to those attained utilizing highly purified, individual isozymes. Therefore, partially purified isozymes were used in many of the experiments reported here. Electrophoresis. The starch gel electrophoretic technique and the tetrazolium method for detecting lactate dehydrogenase activity have been described previously TM. Assays. Enzymatic activity and protein concentration were measured as previously reported 1°. Reagents. All chemicals used in these experiments were of the highest quality commercially available and, with the exception of urea which was recrystallized twice from methanol, were not further purified. RESULTS
Figs. I and 2, respectively, are examples of intraspecific (between shark isozymes) and interspecific (between flying fish and horse isozymes) molecular hybridization. In both of these cases, subunit reassociation was achieved by freezing and thawing the isozyme mixtures in neutral sodium phosphate buffer containing either C1- or urea3,8,1°. This demonstrates that CI-, and the other freeze-thaw hybridizationpromoting substances (Br-, I-, thiocyanate, I-anilinonaphthalene-8-sulfonate)* can be replaced by relatively low concentrations (less than 0.I M) of urea. The characteristics of urea mediated freeze-thaw hybridization are similar to those of halide or thiocyanate-mediated hybridizationS,S,TM. Thus, urea promotes hybridization in the presence of phosphate ions, but, at the same concentrations, is without effect in the absence of phosphate (e.g. in distilled water). Also, Tris blocks hybridization even at urea concentrations many fold greater than required to induced hybridization in the presence of phosphate. Tris stabilizes the enzyme against freeze-thaw mediated destruction. The mechanism by which this is accomplished is unknown. It is not a function of ionic strength. Lactate dehydrogenase is considerably more resistant to freeze-thaw mediated urea " " P r o m o t e r " s u b s t a n c e will be defined o p e r a t i o n a l l y as a n y c h e m i c a l a g e n t w h i c h f u n c t i o n s in c o n j u n c t i o n w i t h o t h e r c h e m i c a l a g e n t s to induce f r e e z e - t h a w r e a s s o c i a t i o n of s u b u n i t s .
Biochim. Biophys. Acta, 1:47 (1967) 45-51
UREA-MEDIATED HYBRIDIZATIONOF L D H
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(+)
ORIGIN
(--) b
c
d
e
f
g
h
Fig. i. Freeze t h a w h y b r i d i z a t i o n of s h a r k l a c t a t e d e h y d r o g e n a s e i s o z y m e s in t h e pre s e nc e of NaC1 or urea. A l i q u o t s of e q u a l e n z y m a t i c a c t i v i t y of t h e m o s t n e g a t i v e l y c h a r g e d i s o z y m e ( L D H - i ) a n d t h e m o s t p o s i t i v e l y c h a r g e d i s o z y m e (LDH-5) were e m p l o y e d . The buffer s y s t e m w a s n e u t r a l o.I M s o d i u m p h o s p h a t e . F r e e z i n g t i m e was 16 h a t - - 2 o °. All s a m p l e s were t h a w e d a t r o o m t e m p e r a t u r e . H y b r i d i z a t i o n was d e t e c t e d b y s t a r c h - g e l e l e c t r o p h o r e s i s a t r o o m t e m p e r a t u r e for 6 h as p r e v i o u s l y d e s c r i b e d l ° . a = L D H - I ; b - LDH-5;c = LDH-I × LDH-5;d = c(LDH-I × LDH-5) + o . o i 5 MNaC1; e = c + o.oo7 M NaC1; f = c + o.oo3 M u r e a ; g = c + o . o o i 5 M u r e a ; h -- c + o.ooo 7 M urea. Due to t h e fact t h a t t h e i s o z y m e s e m p l o y e d in t h i s a n d m o s t of t h e subs e q u e n t e x p e r i m e n t s were c o n t a m i n a t e d w i t h n o n - l a c t a t e d e h y d r o g e n a s e p r o t e i n (see t e xt ), t h e r a t i o s of t h e c o n c e n t r a t i o n of NaC1 or u r e a to e n z y m e p r o t e i n c oul d n o t be c a l c u l a t e d . To p a r t i a l l y c o m p e n s a t e for t h i s deficiency, s e v e r a l d i l u t i o n s of t h e p r o m o t e r s u b s t a n c e s were e m p l o y e d so t h a t a r o u g h e s t i m a t e of t h e i r r e l a t i v e effectiveness could be o b t a i n e d . I t is to be n o t e d t h a t h y b r i d i z a t i o n of s h a r k L D H - I a n d L D H - 5 r e s u l t s in t h e f o r m a t i o n o n l y of 2 i s o z y m e s of i n t e r m e d i a t e m o b i l i t y where, on t h e basis of a s i m p l e b i n o m i a l r e c o m b i n a t i o n , 3 w o u l d be e x p e c t e d . Since i n t e r s p e c i f i c h y b r i d i z a t i o n s t u d i e s h a v e d e m o n s t r a t e d t h a t b o t h s h a r k L D H - I a n d L D H - 5 are h o m o p o l y m e r i c t e t r a m e r s , t h e s e r e s u l t s are difficult to e xpl a i n.
destruction in glass distilled water or Tris than in neutral phosphate buffer even at concentrations of phosphate too low to promote hybridization. Not all isozymic forms of lactate dehydrogenase undergo the same degree of freeze-thaw destruction. At concentrations of enzyme employed in these experiments (approx. o. 7 mg/ml), LDH-5 selectively is destroyed. Selective destruction is enhanced at low phosphate concentrations b y the other promoter substances. In a Tris-HC1 system, the urea concentration m a y be increased to relatively high levels, compared to a phosphate buffer system, with no visible effect on the stability of the enzyme. However, a critical urea concentration (differing for enzymes derived from different sources) exists beyond which LDH-5 is selectively destroyed. Increasing urea concentration above the level Biochim. Biophys. Acta, 147 (1967) 45 51
E. J. MASSARO
48
(+)
0
ORIGIN
(-) a
b
c
d
e
Fig. 2. I n t e r s p e c i f i c f r e e z e - t h a w h y b r i d i z a t i o n of l a c t a t e d e h y d r o g e n a s e i s o z y m e s in t h e p r e s e n c e of NaC1 or urea. A I i q u o t s of e q u a l e n z y m a t i c a c t i v i t y of horse L D H - I a n d fl yi ng fish s k e l e t a l m u s c l e L D H were e m p l o y e d . The buffer s y s t e m was n e u t r a l o . i M s o d i u m p h o s p h a t e . F r e e z i n g t i m e was 15 h a t 20 °. All s a m p l e s were t h a w e d a t room t e m p e r a t u r e . H y b r i d i z a t i o n w a s d e t e c t e d b y s t a r c h - g e l e l e c t r o p h o r e s i s a t r o o m t e m p e r a t u r e for 6 h as p r e v i o u s l y described 1°. a = hors e L D H - I ; b -- flying fish m u s c l e l a c t a t e d e h y d r o g e n a s e ; c -- horse L D H - I × flying fish m u s c l e L D H ; d = horse x fish + o . s M NaC1; e = horse × fish + o . o 6 M urea. The h y b r i d i s o z y m e s are i n d i c a t e d b y s m a l l circles.
effecting LDH-5 destruction results in progressive destruction of the other isozymic forms, utilimately including LDH-I. Preliminary evidence indicates that the order of lability of lactate dehydrogenase isozymes is L D H - 5 > L D H - 4 ~ L D H - 3 LDH-2 > LDH-I. The underlying reason for the increased susceptibility of LDH-5 to freeze-thaw destruction is unknown but appears to stem from structural characteristics common to LDH-5 from m a n y diverse species. Further evidence for a similar mode of action of urea and the halides in the hybridization mechanism has been obtained from complementation experiments. Biochim. Biophys. Acta, 147 (1967) 4 5 - 5 I
UREA-MEDIATED HYBRIDIZATION OF L D H
49
After establishing the minimal urea and NaCI concentration at which unequivocal hybridization could be elicited between shark L D H - I and LDH-5, identical isozyme mixtures were frozen and thawed in the presence of subthreshold concentrations both of urea and NaC1. It was observed that urea and NaC1 act in an additive manner and the hybridization which is obtained is indistinguishable from that produced by either promoter substance alone. Other isozyme combinations give similar results. Finally, F - does not function as a promoter substance 3 and subthreshold concentrations of urea in the presence of fluoride (up to I.O M) do not induce hybridization. As with the interactions of proteins in general, it is not the molar concentrations per se of the components of the hybridization-inducing environment which are of primary significance, but the molar ratios of these substances to enzyme protein. Since, in m a n y of the experiments described, isozyme preparations containing varying quantities (less than IO %) and types of non-lactate dehydrogenase proteins were utilized, no meaningful calculation of these ratios is possible. However, the relative effectiveness of urea in promoting freeze-thaw hybridization in impure enzyme preparation can be estimated. By comparing the minimal molar concentration of NaC1 necessary to induce hybridization in the phosphate system to that of urea, it has been calculated that urea is from two to ten times as effective as NaC1, depending upon the source of the isozymes. Preliminary evidence with highly purified mammalian isozymes indicates that these estimates are reasonably accurate. DISCUSSION
Recombination of lactate dehydrogenase subunits through reversible denaturation at supra-eutectic temperatures employs high concentrations of denaturant to produce dissociation of the tetramer2,4, e. Renaturation is accomplished b y dilution. An analogous cycle of concentration and dilution occurs during freeze-thaw hybridization. Due to the rapid rate of reassociation, the degree of structural perturbation involved in freeze-thaw hybridization must be small. This suggests that only small changes in conformation are necessary to achieve subunit dissociation at supraeutectic temperatures. However, the conditions for achieving these changes are such that high reagent concentrations must be employed. This results in protein denaturation whichis secondary to, although perhaps simultaneous with, subunit dissociation. Freeze-thaw hybridization is known to occur only under a limited number of conditions. The role of each of the components of the hybridization milieu is obscure. Diverse forces are responsible for quaternary structure and the nature of the promoter substances indicates that they act upon different types of forces. However, the same net result is produced. Apparently, selective disruption of certain of these forces will result in reversible dissociation. I t has been suggested that urea m a y function b y an ion-exchange mechanism to disrupt intramolecular bonds 5. In solution, urea m a y exist as a zwitterion with a large, positive, low density, head charge on the nitrogens and an intense negative charge on the oxygen. Under the proper conditions, the low density charge favors binding of the urea "cation" to the surface of macromolecules. This binding would be tenacious and the "cation" would function as a "primer" which dissociates or is displaced b y other functional groups (e.g. water). Halides and other promoter substances m a y function in a similar manner but at different sites. This is Biochim. Biophys. Acta, 147 (1967) 45-5I
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E. J. MASSARO
supported by the observation that the order of effectiveness of the halides in promoting freeze-thaw hybridization (I- > B r - > C1 ) is directly related to the charge density on the ion (I < B r - < C1-). Phosphate, arsenate, and nitrate also might function via a similar ion-exchange mechanism. A voluminous low density charge on the Tris ion, dimethyl sulfoxide, and certain polyhydroxy organic compounds could explain the mechanism by which they inhibit hybridization and protect the enzyme against freeze-thaw mediated urea denaturation. Tile binding of a large number of Tris ions on the surface of the macromolecule would form a "protective" coat against urea and the other promoter substances. A similar argument could be evoked to explain the greater effectiveness of sodium phosphate than potassium phosphate systems in promoting freeze-thaw hybridization a. Because of the larger low density charge on K ~, it might form a "protective" coat over the enzyme which would inhibit the penetration of phosphate and the other promoter substances. The greater susceptability of the LDH-5 type isozymes to freeze-thaw denaturation is difficult to explain since this observation is not limited to homologous isozynles of a single group of closely related organisms. On the contrary, similar results have been observed among the lactate dehydrogenase isozymes of a wide variety of organisms such as the cow, rabbit, horse and shark. Since it has been shown unequivocally that subunit recombination among lactate dehydrogenase isozymes derived from unrelated species is the rule rather than the exception 9, it m a y be that certain structural features of a given lactate dehydrogenase isozymes type are very similar in all species. It would be these features which impart to the molecule the properties characteristic of a particular type of lactate dehydrogenase isozymeL To nlaintain the identity of a given isozyme type, these features would have to be conserved throughout evolution and, in effect, would serve to "dictate" the "acceptable" amino acid substitutions within the basic structure of the molecule which obviously have occurred. The terms "dictate" and "acceptable" are used to emphasize the idea that, should the characteristic structural features of a given isozyme type be modified significantly, the isozyme type would cease to exist or the molecule might cease to function as a lactate dehydrogenase n. Thus, the concept of characteristic structural features implies that certain conformational aspects of a given lactate dehydrogenase isozyme type are universally similar. Such similarities easily account for the selective denaturation of homologous lactate dehydrogenase isozymes. ACKNOWLEDGEMENTS
This investigation was supported by N.S.F. research grant GB 544oX. The technical assistance of Miss S. MOORE is gratefully acknowledged. I am indebted to Professor C. L. MARKERT, Department of Biology, Yale University and to Dr. G. COLACICCO, Department of Biochemistry, Albert Einstein College of Medicine for helpful discussions during the course of this investigation. REFERENCES I S. ANDERSON AND (~. WEBER, Arch. Biochem. Biophys., 116 (1966) 207. 2 0 . P. CHILSON, L. A. COSTELLO AND N. O. I(APLAN, dr. ]!/[ol. Biol., IO (1964) 349. 3 0 . P. CHILSON, L. A. COSTELLO AND N. O. KAPLAN, Biochemistry, 4 (1965) 271.
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4 0 . P. CHILSON, G. B. KITTO, J. PUDLES AND N. O. KAPLAN, J. Biol. Chem., 241 (1966) 2431. 5 G. COLACICCO, Nature, 198 (1963) 583. 6 C. J. EPSTEIN, M. M. CARTER AND R. F. GOLDBERGER, Biochim. Biophys. Acta, 92 (1964) 391. 7 N. O. KAPLAN AND M. M. CIOTTI, Ann. N . Y . Acad. Sci., 94 (1961) 7Ol. 8 C. L. 1ViARKERT, Science, 14o (1963) 1329. 9 C. L. MARKERT, Harvey Lectures Ser., 59 (1965) 187. IO C. L. MARKERT AND E. J. MASSARO, Arch. Biochem. Biophys., 115 (1966) 417 . I1 K. E. NEET AND D. E. KOSI-ILAND, Jr., Proc. Natl. Acad. Sci. U.S., 56 (1966) I6o6.
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