Function of zinc in horse liver alcohol dehydrogenase

ARCHIVES

OF

BIOCHEMISTRY

Function

AND

of Zinc

HANNAH

BIOPHYSICS

119,

in Horse

552-559

(1967)

Liver Alcohol

L. OPPENHEIMER, ROBERT AND ROBERT H. MCKAY

Department of Biochemistry School of Medicine,

Dehydrogenase’ W. GREEN,

and Biophysics,

University

Honolulu,

Hawaii 96822

of Hawaii

Received September 6, 1966 The zinc content of horse liver alcohol dehydrogenase (LADH) was determined by atomic absorption spectrophotometry and found to be four atoms of zinc per molecule of enzyme. LADH containing various amounts of zinc was prepared, and the zinc content was related to the enzyme’s sulfhydryl group lability, conformation, catalytic activity, and stoichiometry of coenzyme binding. Ultraviolet difference spectra revealed that a conformational change appears upon removal of zinc from LADH. This change takes place prior to sulfhydryl group oxidation to disulfide bonds, which occurs readily in LADH containing less than four atoms of zinc. The specific activity of LADH decreases as zinc is removed from the enzyme even when all sulfhydryl groups remain intact. Starch gel electrophoresis of LADH suggests that only the four zinc LADH species retains catalytic activity. The relationship between zinc loss and activity is nonlinear. However, a linear relationship was observed between LADH zinc content and stoichiometry of coenzyme binding.

liver Horse alcohol dehydrogenase (LADH)” (E.C. 1.1.1.1 alcohol; NAD oxidoreductase, equine liver) contains zinc, which is essential for its catalytic activity (1; see also reviews, 2-5). Recently Akeson demonstrated that the zinc content of LADH is four atoms of zinc per molecule, and suggested that two zinc atoms are enzymically functional while the other two may contribute to maintaining the tertiary structure of the protein (6). This paper describes methods for preparing reasonably stable low r This work was supported by grant GM-11632 from the National Institutes of Health and by Research Career Development Award 5-K3-GM8517 from the National Institutes of General Medical Science to R. H. McKay. 2 Predoctoral trainee of the National Institutes of Health (grant GM-1039). 3Abbreviations used: LADH, horse liver alcohol dehydrogenese; NAD and NADH2, oxidized and reduced forms of nicotinamide adenine dinucleotide; PCMB, p-chloromercuribenzoate; DTT, dithiothreitol; ADPR, adenosine 5’ - diphosphoribose; Tris, 2-amino-2-hydroxymethyl-propane-l $diol.

zinc enzyme and correlates LADH zinc content to enzyme structure, specific activity, and coenzyme binding. A preliminary report of this work has appeared (7). EXPERIMENTAL

PROCEDURES

MATERIALS Crystalline LADH, prepared by Boehringer and Sons, was obtained from Calbiochem, Los Angeles, California. LADH Lots 45456 and 52223 were used in these experiments. NADHz, Lot 2236, was purchased from P-L Biochemicals, Inc., Milwaukee, Wisconsin. ADPR, Lot 43B-673 was from Sigma Chemical Company, St. Louis, Missouri; and NAD, DTT, PCMB, and Tris were from Calbiochem. Ethanol, 95%, was obtained from Commercial Solvents Corporation, Agnew, California and used without further purification. All other chemicals were reagent grade. Spectrographically pure metallic zinc, purchased from Johnson Matthey Company, Ltd., London, England, was used for zinc standard curves. Visking 18/32 seamless cellulose tubing from Visking Corporation, Chicago, Illinois was used for dialysis. Apparatus. A Radiometer pH stat (TTT lb)

552

ZINC

IN

HORSE

LIVER

ALCOHOL

was used for pH determinations. For absorbance readings, a Zeiss PM&-II spectrophotometer was used. A Gary model 14 spectrophotometer was used for enzyme activity assays and for difference spectra. The Zeiss spectrophotometer was adapted for atomic absorption measurements (8). The emission source was a brass hollow cathode (Westinghouse WL 22607). Samples were aspirated directly into a horizontal ceramic tube through a Beckman hydrogen-air burner.

METHODS Preparation of LADH and low zinc LADH. Ten ml of crystalline LADH suspension (100 mg) was dialyzed against 2 X 500 ml of 0.05 Tris-HCl buffer, pH 7.5, for 18 hours at 4”. Any insoluble material remaining was removed by filtration through a Millipore filter, 0.8 p pore size. This preparation contained four zinc atoms per molecule LADH. Five ml of 4-zinc LADH was adjusted to pH 5 with acetic acid and dialyzed against 1 liter of 0.05 M sodium succinate buffer, pH 5.0,4”. Dialysis tubing was pre-soaked in deionized water. The anaerobic dialysis was performed in a closed container with an inlet and an outlet for bubbling nitrogen through the dialyzate. The half time for zinc removal under these conditions is 6.5 hours. Zinc removal jrom LADH (kinetic study). Dialysis was performed with a diffusion cell based on one designed by Craig and Konigsberg (9). Dialysis tubing was cleaned by the method of Hughes and Klotz (10). One ml of LADH containing approximately 7.5 mg was adjusted to pH 5.0 with acetic acid. Three-ml aliquots of 0.05 M sodium succinate, pH 5.0, buffer were changed every hour for 24 hours. The film of dialyzing solution was approximately 0.15 mm thick; the dialyzing surface was 24 cm2. Zinc in the dialyzate and in the enzyme before and after dialysis was determined by atomic absorption spectrophotometry. A buffer blank control diaIysis was run and subtracted from the enzyme dialysate samples. Enzyme and coenzyme concentration determination. LADH concentration was determined spectrophotometrically at 280 rnp using an absorptivity of 0.42 mg-1 cm%, and molar concentrations are based on a molecular weight of 84,000 (11). The concentration of NADHz was determined by measuring its absorbance at 340 mp using a molar absorptivity of 6.22 X lo3 (12). The NADHz was dissolved in 0.05 M Tris-HCl, pH 7.5. For NAD, a molar absorptivity of 18.0 X lo3 at 260 rnk and pH 7 (13) was used to calculate the concentration. Enzyme assay. LADH specific activity was de-

DEHYDROGENASE

5.53

termined at pH 8.8 in Tris-HCl buffer, p = 0.1, in the presence of 1.7 X lO+ M NAD and 1.6 X 10m2 M ethanol. Activity was determined within 1 minute after addition of enzyme to the assay cuvette. The enzyme concentration in the assay cuvette was approximately 1.5 pg/ml (1.8 X IO-+ M). The average molecular activity was 750 moles NAD reduced per mole of protein per minute at 25”, pH 8.8. The temperature dependence of molecular activity was approximately 26 molecular activity units/‘% over the range 16-32”. Zinc analysis. The procedure described by Fuwa et al. (14) was used to determine the zinc content of LADH preparations. Enzyme samples, diluted with deionized water to concentrations containing between 0.1 and 0.4 pg zinc/ml, were run in triplicate. Buffer or reagent blanks were subtracted. National Bureau of Standards standard sample 157-a was analyzed to check the accuracy of the standard curve. Sulfhydryl group analysis. The sulfhydryl titer of LADH preparations wm measured with PCMB by spectrophotometric titration (15, 16), except that the assays were performed in 0.05 M glycineHCl buffer, pH 3.0, since low zinc-LBDH is more soluble at acidic pH. Glutathione, used as a standard, reacted rapidly and gave the expected equivalence point at this pH. Preparation of samples for DTT reaction. Low zinc-LADH in 0.05 M sodium succinate, pH 5.0, was adjusted to pH 7.5 by adding an equal volume of 0.05 M Tris. It was stored at 4“ for several days to allow the sulfhydryl titer to decrease. A 4.0-ml aliquot of the enzyme was added to 2.40 pm urea, buffer, pH 7.5, and then 0.65 ml of 0.05 M Tris-HCl was added, bringing the urea concentration to 6.25 M and the enzyme concentration to approximately 3 X 10e5 M. Any turbidity was removed by filtering the sample through a 0.8 p pore size Millipore filter. The 4 zinc-LADH control was prepared by adding 2.0 ml enzyme solution to 2.40 gm urea and diluting with 2.65 ml of 0.05 1~ Tris-HCl buffer, pH 7.5. A 2.5 X 1O-4 M cystine solution in 0.05 M Tris-HCl buffer, pH 7.5, was diluted with urea and buffer in the same way as 4 zinc-LA41)~I. The conditions for reduction of disulfide horlds with DTT were those used by Cleland (17). Difference spectra measurement. High US. low zinc LADH: To 2.0 ml of 4 zinc-LADH in 0.05 M Tris HCl, pH 7.5, was added 2.0 ml of 0.03 M sodium succinate, pH 5.0. The resulting solution had a pH of 5.2 and a protein concentration of approximately 3.5 mg/ml. This solution was allowed to stand at 4” under nitrogen for 24-48 hours. The zinc loss was estimated by the decrease in specific activity. Immediately before running the differ-

554

OPPENHEIMER,

GREEN,

ence spectrum, the 4 zinc-LADH stock was diluted 1:l with 0.05 M sodium succinate, pH 5.0, and a difference spectrum was obtained from 400 to 250 w. LADH and DTT vs. LADH: To 3.0 ml enzyme (approximately 2.5 mg/ml at pH 7.5) was added 0.06 ml of a 3 X 10e2 M solution of DTT. To a second R.O-ml aliquot, 0.06 ml water was added. The same pipettes were used for each pair of samples in order to minimize concentration discrepancies. Difference spectra (350-250 rnb) were recorded at intervals between 15 minutes and 1 hour. The difference spectra of cystine and DTT vs. cystine were obtained identically to those of the enzyme. All samples were assayed in 6.25 M urea. The urea prevented precipitation of oxidized low zinc-LADH which is unstable at 23” and pH 7.5, the conditions required for the DTT reaction. Reagent blank difference spectra (buffer + urea + DTT vs. buffer + urea) were checked for all samples. Starch gel electrophoresis. Starch gel electrophoresis was carried out according to the method of Smithies (18) using 0.05 M sodium succinate buffer, pH 5.0. Connaught starch, 62 gm, was mixed with 500 ml of buffer to form the gel. The runs were done on the apparatus of Ashton and Braden (25) at 4” for 2 hours; a power supply voltage of 300 V and a current of 100 mA were used across 6 starch gel strips which were 59 mm wide, 3 mm thick, and 252 mm long. Approximately 0.7 mg of LADH was applied to each gel strip from a piece of filter paper placed in the gel. Protein bands were observed by staining with nigrosine. For quantitation, bands from unstained gel strips were cut out, gently homogenized with a glass rod in a minimum volume of 0.05 M Tris buffer, pH 8.0, and then centrifuged in the cold. The supernatant fluids were assayed for specific activity and zinc content. The volume of the supernatant fraction was corrected for the liquid volume of the gel.

AND MCKAY

The amount of ADPR binding to LADH was measured by adding increasing amounts of 5 X lo-

M ADPR

t0

o.kkd

EdiCpOti

Of LADH

(7.5

mg/

ml) at pH 8.4. The enzyme-coenzyme complex was precipitated by the addition of 1.5 ml of 95y0 ethanol, and the supernatant fraction was separated by centrifugation. The extent of binding was calculated after spectrophotometric determination of the concentration of ADPR remaining in the supernatant fraction. E’~‘“” of ADPR is 16 X lo3 (19a). RESULTS

LADH

zinc content and enxymic activity.

The zinc content of LADH was found to be 3.9 f 0.1 atoms of zinc per molecule of enzyme, in agreement with the recent observation of Akeson (6). Zinc is removed from LADH at a first-order rate until 25 % of the zinc remains. Removal of the final zinc deviates from the first-order rate of the first 75 % zinc and appears to follow second order kinetics (see Fig. 1). As zinc is removed from LADH, there is an accompanying loss in specific activity. This decrease in activity is nonlinear (Fig. 2). The relationship between specific activity and zinc content is maintained over a tenfold range of coenzyme and substrate concentration. The enzymic activity was determined at pH 8.8 immediately after removing the sample from the pH 5.0 anaerobic dialysis. When LADH is zinc-free, no enzymic activity is observed. Specific activity was found to be a sensitive measure of zinc content. LADH zinc content and ~~hy~ryZ group titer. Four zinc-LADH has 27.5 f 0.8

sulfhydryl groups per molecule, a value based on 14 determinations. The sulfhydryl titer of low zinc-LADH remains unchanged coenzyme bound per mole of LADH was deterat 28 groups per molecule when measured mined as function of zinc at pH 8.4 and 9.0 using within a few hours after the pH 5.0 anaerobic the spectrophotometric method of Ehrenberg and dialysis. However, a decrease of sulfhydryl Dalziel (11). Two ml of LADH, approximately 7 groups was observed when the low zincmg/ml in either 0.05 M Tris-HCl buffer, pH 7.5, or LADH preparations were exposed to air for 0.05 M sodium succinate buffer, pH 5.0, were diseveral days (Table I). This loss of sulfhydryl luted to 6.5 ml with 4.5 ml of 0.1 M glycine-NaOH buffer, pH 10, or Tris-HCl buffer, or = 0.1, pH 8.8. groups is faster at pH 7.5 than at pH 5.0. The LADH concentration during these exThe final pH’s were 9.0 and 8.4, respectively. The periments was between 3.5 and 8.3 X W5 M. assays were carried out at 12”, a temperature To determine whether the decrease in where low zinc-LADH remains soluble at these pH’s. Absorbancies were measured at 355 and 310 sulfhydryl titer was due to conformational change resulting in the burying of sulfhydryl 9. Determination NADH2, LADH

of stoichiometry: LADH + + ADPR. The maximum moles of

ZINC

IN

HORSE

LIVER

ALCOHOL

DEHYDROGENASE

. .06

1 0

IO

DIALYSIS

TIME

m

0

20

(HOURS)

1. First- and second-order plots of percentage of zinc bound to LADH vs. dialysis time at pH 5.0, 4’. FIG.

TABLE DECREASE

100

: 50

Ir!

IL

l. ’

6 ; 90

0100

IN

Low

ZINC-LADHa

z;i 100 5 w a

5 F Y

I

IN SULFHYDHYLGROUPS

75

% ZINC

50

25

l\ a\. 0

REMAINING

2. Plot of percentage of activity remaining vs. percentage of zinc remaining. 100% activity represents a molecular activity of 750 moles NAD per minute per mole of enzyme at pH 8.8 and 25”. 100% zinc represents 3.9 f 0.1 atoms per molecule LADH. FIG.

groups previously accessible to PCMB, LADH preparations were assayed for sulfhydryl groups in 8 M urea at pH 3.0. It was assumed that denaturation of the enzyme in 8 M urea would expose any buried sulfhydryl groups to PCMB. The sulfhydryl titer of native LADH and low zinc-LADH was unchanged when measured in 8 M urea as was that of the glutathione standard. Upon prolonged standing in S ELIurea, a decrease in

100 67.5 62.5 67.5 67.5 62.5

0

7.5

21 1 4 8 0 4

7.5 7.5 7.5 7.5 5.0 5.0

27.2 f 0.8 27.2 22.8 20.0 16.6 27.2 26.4

a LADE containing less than 507, Zn precipitates at pH 7.5.

sulfhydryl groups was observed in all preparations. Since the sulfhydryl group loss in low zincLADH could not be attributed to buried unreactive groups, the possibility that oxidation to disulfide bonds had occurred was examined. DTT is capable of reducing disulfides quantitatively, and the number of disulfide bonds reduced can be estimated from the absorbance of the oxidized DTT formed. The molar absorptivity of oxidized DTT is 265 at 280 rnM (17). Fig&e 3 shows that LADH (100 % Zn) contains no disulfide bonds, while LADH (43 % Zn) stored for 3 days at pH 7.5 and

556

OPPENHEIMER,

GREEN,

4’, has approximately 5 disulfide bonds per molecule. The sulfhydryl titer of LADH (43% Zn) indicated a loss of 12 sulfhydryl groups, in reasonably good agreement with the DTT data. A difference spectrum of the model compound, cystine + DTT vs. cystine (Fig. 3), is identical in shape to that of the low sulfhydryl-LADH. This provides further evidence that some cysteines of LADH become cystine residues when the low zinc enzyme preparation is exposed to oxygen. LADH zinc content and conformation. Since spectral shifts are an indication of conformational changes in proteins, the technique of ultraviolet difference spectroscopy was used to detect the effect of zinc removal on LADH structure. In Fig. 4, two characteristic difference spectra of high zinc LADH vs. freshly prepared low zinc LADH reveal a peak in the 300-285 rnp region and a trough in the 275-250 rnp region. The trough increases in magnitude with decreasing LADH zinc content. Spectrophotometric titration with PCMB showed that both of the low zinc-LADH preparations retained their 28 sulfhydryl groups intact. Therefore, the difference spec-

WAVELENGTH

(mp)

FIG. 3. Ultraviolet difference spectra in 0.05 M Tris-sodium succinate buffer, pH 7.5, 6.25 M urea, 25”. DTT concentration is 5.75 X 10-* M. ET”” of oxidized DTT is 265. (... .) 100% zinc-LADH + DTT vs. lOO$&zinc-LADH. 3.75 X 1OWM protein, 26 sulfhydryl groups per molecule LADH, 30-minute reaction time with DTT. (- - -) 43% zinc-LADH + DTT vs. 43% zinc-LADH. 3.2 X lO-& M protein (stored at 4”, pH 7.5, for 3 days), 14 sulfhydryl groups per molecule LAPH, 30minute reaction time with DTT. (--) Cystine + DTT vs. cystine. 2.5 X 1CF M cystine, 15-minute reaction time with DTT.

AND MCKAY

240

260

260

300

WAVELENGTH

320

340

(mp)

FIG. 4. Ultraviolet difference spectra in 0.05 M Tris-HCl-sodium succinate buffer, pH 5.2, 25’. 3.2 X lO+ M protein, 28 sulfhydryl groups per molecule LADH. (--) 100% zinc LADH vs. 7570 zinc-LADH. (- - -) lOO’% zinc-LADH vs. 60% zinc-LADH .

NATIVE

LADH

LOW

Zn - LADH -ORIGIN -BAND 2

(4

-WIND I w % SPECIFIC ACTIVITY INrrIAL BAND I BAND 2

ENZYME NATIVE LOW

LAD” Zn-

LAD,,

100

72

94

62

NONE

FIG. 5. Starch gel electrophoresis

x INIT, I. FiEwN 4c 0 65 67

of native and

low zinc LADH at pH 5.0.

tra are not the result of disulfide bond formation in the low zinc enzymes. The concentration, pH, and ionic strength of high and low zinc LADH pairs were identical, so the spectral shifts observed can be attributed to a conformational change in the protein arising from the loss of zinc. Starch gel eEeetrophore&. Starch gel electrophoresis at pH 5.0 of the native and lowzinc enzyme preparations suggests that the activity measured in the low zinc material is due to residual native LADH. Native LADH produced one protein band after starch gel electrophoresis, while the low zinc preparations were resolved into a major and minor protein band, the major band having a mobility identical to the native control (Fig. 5). The specific activity of the major band (band 1) of the low zinc enzyme increased almost to the level of the native control. Band 2 was inactive and contained no zinc. Approximately 80% of the protein could be recovered after electrophoresis. Because

ZINC IN HORSE LIVER

ALCOHOL

DEHYDROGENASE

557

The enzyme concentration used in these stoichiometry determinations was much larger than the largest dissociation constant of the coenzyme (K = 2.7-3.1 X lo--’ M at pH 7) (19, 27), so the necessary conditions of stoichiometric addition were maintained (20).

100 1

DISCUSSION

0-

2

0

FIG. 6. Plot of percentage of zinc remaining vs.

moles NADHz and ADPR per mole LADH. Zinc content was determined by atomic absorption spectroscopy, NADHz binding by spectrophotometric titration at pH 8.4 and 9.0, and ADPR binding by spectrophotometry after precipitation of the complex at pH 8.4. [NADH]/[LADH] = 0; [ADPRI/ILADH] = 0.

it was necessary to carry out electrophoresis at pH 5 in order to avoid precipitation of the low zinc enzyme, some zinc and activity were lost in the native enzyme control. The possibility is not excluded that band 1 includes low zinc protein which may or may not contribute to the activity observed. No material was left at the origin when electrophoresis was done immediately after zinc removal. However, if about 2 hours elapsed prior to electrophoresis, some aggregate was observed at the origin.4 LADH zinc content and stoichiometry of coenzyme binding. There is a linear relationship between atoms zinc per molecule LADH and moles NADHz bound per mole LADH (Fig. 6). Each point represents an average of four stoichiometry determinations made at two wavelengths and two pHs. The protein concentration was between 11 and 28 I.IM and the aliquots of coenzyme added covered the range from 0 to 110 PM NADH2. The coenzyme analogue, ADPR, does not differ from NADHz in the extent of binding to low zinc LADH (Fig. 6). ADPR stoichiometry measurements were made with 15-22 pl\r LADH and O-160 PM ADPR. 4 Preliminary data from sedimentation velocity studies show aggregation in LADH preparations containing less than 50’% zinc (26).

The experiments described were designed to clarify the functional significance of zinc in LADH and to attempt to distinguish between effects which are specifically related to the enzyme’s binding or catalytic sites and effects which have a less specific structural influence on the protein molecule. Zinc analyses by atomic absorption spectrophotometry and sulfhydryl groups assay by spectrophotometric titration with PCMB demonstrated that LADH contains 3.9 f 0.1 atoms of zinc and 27.5 f 0.8 sulfhydryl groups per molecule. These values confirm the LADH zinc analyses of Akeson (6) and are in agreement with the sulfhydryl data of Witter (21). When zinc is removed from LADH by anaerobic dialysis at pH 5, the sulfhydryl titer remains unchanged. However, a decrease of sulfhydryl groups was observed when the low zinc enzyme preparations were exposed to air (Table I). Native LADH has remarkably stable sulfhydryl groups. Sulfhydryl group loss occurs as soon as the zinc content is lowered and reducing conditions are not maintained. This loss of sulfhydryl groups is greater at pH 7.5 than at pH 5 as might be expected if oxidation to disulfide were the cause. The sulfhydryl titer did not increase when the assay was performed in 8 M urea, suggesting that all the sulfhydryl groups in the molecule were accessible to PCMB. DTT was used to determine the presence of disulfide bonds in LADH. DTT reduces disulfide bonds quantitatively, and the number of disulfide bonds reduced can be estimated from the absorbance of oxidized DTT. By measuring difference spectra, the contribution of protein absorbance is cancelled out and the relatively small absorbance of oxidized DTT can be determined (Fig. 3). Native LADH contains no disulfide

558

OPPENHEIMER,

GREEN,

bonds while low zinc-LADH contains vary ing amounts of disulfide bonds. The loss of zinc must therefore change the enzyme structure sufficiently to enable sulfhydryl groups to react with each other. Steric restrictions probably prevent this in the native enzyme (22). Since spectral shifts are a sensitive indication of conformational changes in proteins (23, 24), the technique of ultraviolet difference spectroscopy was also used to detect the effect of zinc removal on LADH structure. The difference spectra of native LADH vs. low zinc-LADH (Fig. 4) are typical of perturbations arising from slight environmental changes near the protein’s chromophoric groups. Sulfhydryl group assay performed immediately after ‘measuring the difference spectra showed that all 28 sulfhydryl groups were intact, so these difference spectra cannot be attributed to disulfide bond formation in the low zinc enzymes. Since the concentration, pH, and ionic strength of high and low zinc enzyme pairs were identical, the spectral shifts observed are most likely the results of conformational changes in the protein due to loss of zinc. Dialysis studies at pH 5 (Fig. 1) revealed that zinc is removed from LADH at a firstorder rate until an average of 25 % zinc per molecule remains. This result implies that either the zinc atoms are equivalently bound to the enzyme or that the removal of the first zinc atom is rate-determining. The apparent change in mechanism for removal of the final zinc atoms may be due to aggregation of LADH. The relationship between zinc content and LADH activity is not a linear one, as might be anticipated (Fig. 2). The nonlinear nature of the curve may indicate that the 4 zinc atoms are nonequivalent in their influence on catalytic activity. Starch gel electrophoresis experiments suggest that the activity measured in the low zinc enzyme preparations is due to residual native LADH. The complete lack of activity and zinc in band 2 and the agreement in the specific activity of band 1 in the native and low zinc preparations support the idea that the presence of all four zinc atoms is critical for activit,y (Fig. 5). Attempts to

AND

MCKAY

isolate an active, low zinc LADH have been unsuccessful so far. The existence of such a species could explain the zinc-activity relationship shown in Fig. 2. The stoichiometry of NADHS and ADPR binding is directly proportional to the LADH zinc content. Both the coenzyme, NADH2, and the coenzyme analogue, ADPR, bind to the same extent. ADPR lacks the nicotinamide portion of NADHS and unlike NADHz binds to LADH in the presence of the zinc chelating reagent 1, IO-phenanthroline (19a). Since bothADPR and NADHz binding are affected by zinc removal from LADH, a general disintegration of the coenzyme binding site seems likely. The linear relationship between coenzyme binding and enzyme zinc content suggests that the zinc atoms although not necessarily binding directly to the coenzyme, are equivalent in their effect on coenzyme binding. Since an LADH preparation containing 50 % zinc still binds 50 % of the coenzyme but has only 30% catalytic activity, it appears that loss of coenzyme binding capacity does not fully account for the activity decrease observed. The data presented emphasize the role of zinc in maintaining LADH’s catalytically active structure. Zinc removal results in conformational changes followed by oxidation of formerly stable sulfhydryl groups to disulfide bonds. A disruption of both catalytic and coenzyme binding sites also appears to accompany the loss of zinc from LADH. However, catalytic and coenzyme binding properties are affected in different ways. ACKNOWLEDGMENT The authors wish to thank Mr. Henry for skilful technical assistance.

Funasaki

REFERENCES 1. VALLEE, B. L., AND HOCH, F. L., J. Biol. Chem. 336, 185 (1957). 2. VALLEE, B. L., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. MyrbLck, eds.), 2nd edition, Vol. 3, p. 225. Academic Press, New York (1960). 3. SUND, H., AND THEORELL, H., in “The Enzymes” (P. D. Boyer, H. Lardy, and K. MyrbBck, eds.), Vol. 7, p. 25. Academic Press, New York (1963).

ZINC

IN HORSE

LIVER

ALCOHOL

4. MCKINLEY-MCKEE, J. S., Prog. Biophys. 14, 225 (1964) . 5. Sund, H., 2. Naturwiss, Med. Grundlagenjorsch. 2(3), 284 (1965). 6. AKESON, A., Biochem. Biophys. Res. Commun. 17, 211 (1964). 7. OPPENHEIMER, H. L., .~ND MCKAY, R. H., Federation Proc. 26, 585 (1966). 8. FUWA, K., AND VALLEE, B. L., Anal. Chem. 35, 942 (1963). 9. CRAIG, L. C., AND KONIGSBERG, W., J. Phys. Chem. 65, 166 (1961). 10. HUGHES, T. R., AND KLOTZ, I. M., in “Methods of Biochemical Analysis” (D. Glick, ed.), p. 265. Wiley (Interscience), New York (1956). 11. EHRENBERG, A., AND D.~LZIEL, K., Acta Chem. &and. 12, 465 (1958). 12. HORECKER, B. L., AND KORNBERG, A., J. Biol. Chem. 176, 385 (1948). 13. KORNBERG, A., AND PRICER, W. E., in “Biochemical Preparations” (E. E. Snell, ed.), Vol. 3, p. 23. Wiley, New York (1953). 14. FUU’A, K., PULIDO, P., MCKAY, R., AND VALLEE, B. L., Anal. Chem. 36, 2407 (1964).

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15. BOYER, P. D., J. Am. Chem. Sot. 76, 4331 (1954). 16. SWENSON, A. D., AND BOYER, P. D., J. Am. Chem. Sot. 79, 2174 (1957). Ii’. CLELBND, W. W., Biochemistry 3, 480 (1964). 18. SMITHIES, O., Biochem. J. 61, 629 (1955). 19. THEORELL, H., AND MCKINLEY-MCKEE, J. S., Acta Chem. Stand. 16, 1811 (1961). lga. YONET.INI, T., ilCta Chem. &and. 17, SQ6 (1963). 20. WEBER, G., in “Molecular Biophysics” (B. Pullman and M. Weissbluth, eds.), p. 369. Academic Press, New York (1965). 21. WITTER, A., Acta Chem. Scan& 14, 1717 (1960). 22. CECIL, R., in “The Proteins” (H. Neurath, ed.), 2nd edition, Vol. 1, p. 379. Academic Press, New York (1963). Structure,” p. 217. 23. SCHERAGA, H., “Protein Academic Press, New York (1961). 24. YANKEELOV, J. A. JR., AND KOSHLAND, D. E., J. Viol. Chem. 240, 1593 (1965). 25. ASHTON, G. C., .IND BRADEN, A. W. H., Au&. J. Biol. Sci. 14, 248 (1961). 26. GREEN, R. W., Unpublished results. 27. DALZIEL, K., Acta Chem. Stand. 17, S27 (1963).