Hybrids of chemical derivatives of Escherichia coli alkaline phosphatase

Biochimica et Biophysiea Acta, 412 (1975) 262-272

© Elsevier Scientific Publishing Company, Amsterdam --Printed in The Netherlands BBA 37202 HYBRIDS OF C H E M I C A L DERIVATIVES OF E S C H E R I C H I A C O L I ALKALINE PHOSPHATASE

EDWARD MEIGHEN and RANDOLPH YUE Department of Biochemistry, McGill University, Montreal, Quebec (Canada)

(Received April 14th, 1975)

SUMMARY The activities of hybrid dimers of alkaline phosphatase containing two chemically modified subunits have been investigated. One hybrid species was prepared by dissociation and reconstitution of a mixture of two variants produced by chemical modification of the native enzyme with succinic anhydride and tetranitromethane, respectively. The succinyl-nitrotyrosyl hybrid was separated from the other members of the hybrid set by DEAE-Sephadex chromatography and then converted to a succinyl-aminotyrosyl hybrid by reduction of the modified tyrosine residues with sodium dithionite. A comparison of the activities of these two hybrids with the activities of the succinyl, nitrotyrosyl and aminotyrosyl derivatives has shown that either the subunits of alkaline phosphatase function independently or if the subunits turnover alternately in a reciprocating mechanism, then the intrinsic activity of each subunit must be strongly dependent on its partner subunit.

INTRODUCTION Alkaline phosphatase from Escherichia coli is a dimeric enzyme that contains two identical subunits [1]. This enzyme catalyzes the transfer of phosphate from phosphate esters to water or acceptor alcohols [2]. The mechanism consists of two basic steps; (a) the formation of a phosphorylated enzyme intermediate with the release of alcohol and (b) the dephosphorylation of this intermediate. A number of studies have indicated that only one subunit in alkaline phosphatase may function at any given time. Pre-steady state kinetics under conditions where the rate-limiting step occurs subsequent to the formation of the phosphorylated enzyme intermediate have given results showing the release of one mol of alcohol per mol of dimeric enzyme [3, 4]. The covalent incorporation of phosphate into the enzyme at acidic pH has also only given a single site [5, 6]. Binding studies with phosphate have provided evidence for two phosphate sites with substantially different affinities [7, 8]. From these results it has been suggested that alkaline phosphatase may exhibit negative allosteric behaviour permitting only one active site to function at a given time. A flip-flop or reciprocating subunit mechanism has been proposed by Lazdunski et al. [9] to explain this behaviour. The mechanism is basically a negative allosteric mechanism in which the subunits alternately turn over in the

263 catalytic reaction. A similar mechanism that differs only with respect to the ratelimiting step has also been suggested by Halford [10]. Recently, Bloch and Schlesinger [11] have shown that 1.4 to 1.6 tool of pnitrophenol are released in a pre-steady state burst from p-nitrophenyl phosphate if alkaline phosphatase is purged of endogenous phosphate. These workers have seriously questioned the proposal of negative allosteric behaviour in alkaline phosphatase and have suggested the subunits function independently at least in the presteady state. Furthermore, recent work by the same authors [12] with genetic variants of alkaline phosphatase has also indicated that the subunits may function independently. The independent and reciprocating subunit models for alkaline phosphatase cannot be directly distinguished by steady state kinetics on the native enzyme, since both models predict Michaelis-Menten behaviour. However, steady-state kinetic studies on hybrids of alkaline phosphatase containing two different subunits may distinguish between these models, since both subunits must turnover in the flip-flop mechanism, whereas this is not the case for the independent subunit mechanism. The preparation of such hybrid species requires two variants of alkaline phosphatase not only with different catalytic properties, but that also differ in some property such as electrophoretic mobility so that the hybrid species can be separated from the variants. An electrophoretic variant of a native enzyme can be prepared by limited modification with succinic anhydride to give a species with the same quaternary structure but a different charge than the native enzyme [13]. However, a second variant with different kinetic properties than the succinyl derivative is also required. Such a variant can be prepared by reaction of tetranitromethane with alkaline phosphatase followed by reduction of the nitrotyrosyl residues with Na2S204 [14]. This variant differs in both activity and electrophoretic mobility from the succinyl variant. The investigation of the kinetic properties of a hybrid of these two variants is the purpose of this report. A preliminary account of part of this work has been recently reviewed [15]. METHODS

Enzyme assays. Enzyme activities were measured in 1 ml of 1.0 M Tris/chloride, pH 8.0 (vv) or 1.0 ml of 0.01 M Tris/chloride, 1 M NaCI, pH 8.0 (vH), containing 10 -3 M p-nitrophenyl phosphate. The release ofp-nitrophenol was followed spectrophotometrically at 400 nm as a function of time. A unit of activity is defined as the production of 1 #tool ofp-nitrophenol per rain at 24 °C based on a molar extinction coefficient of 1.7.104 for p-nitrophenol at pH 8.0. The specific activity is given as the units of activity in 1.0 M Tris/chloride, pH 8.0, per mg of alkaline phosphatase. Protein concentrations were based on a specific extinction coefficient of 0.72 (0.1 ~ , 1 cm) at 278 nm [16]. Enzyme purification. E. coli, strain C-90, from the E. coli Genetic Stock Centre, Dept. of Microbiology, Yale University, was grown in the peptone/glucose/salts medium of Levinthal et al. [17] containing 5.10 -5 M inosine [18]. Ten 2-1 flasks containing 1 1 of medium were inoculated with a total of 50 ml of culture (As90 ---- 1.5) and incubated on a rotary shaker at 37 ° C for 20 h until the activity reached 0.8 units/ ml for the cell suspension. The cells were harvested by centrifugation and lysed at

264 room temperature by suspension in 1 1 of 0.033 M Tris/chloride, pH 8.0, 20 70 sucrose, followed by the addition of EDTA and lysozyme [16]. The supernatant, containing 10 000 units of activity, was equilibrated with 40 g of DEAE-cellulose (DEAE-32, Whatman) at 4 °C for 2 h, NaCI added to a final concentration of 0.2 M, and the DEAE-cellulose removed by centrifugation. The supernatant was then concentrated in dialysis bags covered with dry Aquacide (Calbiochem) followed by dialysis at 4 °C into 0.01 M Tris/chloride, 0.05 M NaC1, 3 mM mercaptoethanol, 2 mM ZnC12, 1 mM MgSO4, pH 7.4. The sample was applied to a 2.5 cm × 50 cm DEAE-Sephadex A-50 (Pharmacia) column and eluted with 600 ml of a linear gradient from 0.05 to 0.20 M NaC1 in 0.01 M Tris/chloride, pH 7.4, containing 3 mM mercaptoethanol, 2 mM ZnCI2 and 1 mM MgSO4. The peak of activity (40-50 units/mg) was precipitated by dialysis against saturated ammonium sulfate, pH 7.0, and stored as an ammonium sulfate suspension at 4 °C. Cellulose acetate electrophoresis. Electrophoresis was conducted in 0.04 M Na/K phosphate, pH 7.0, on cellulose polyacetate strips (Gelman Sepraphore III) in a Microzone electrophoresis cell (Model R-101, Beckman-Spinco). A voltage of 200 V was applied for 15 min. The protein was fixed and stained by immersion of the membrane in a solution of Ponceau S in trichloroacetic acid and sulfosalicylic acid (Beckman-Spinco) for 7 min and then rinsed in 5 70 acetic acid. A darker stain was obtained by further immersion in 0.002 700nigrosin in 2 70 acetic acid for several hours. Alternatively, the enzyme activity was detected by a histological stain using a-napthyl acid phosphate and fast red violet salts as described by Hoffman and Wilhelm [19]. The membrane, after rinsing in 5 70 acetic acid, was dried and stored at room temperature. Sedimentation velocity experiments. Sedimentation velocity experiments were performed in a Beckman Spinco Model E analytical ultra-centrifuge using the Schlieren optical system and 12-mm double-sector cells with aluminium filled, epoxy centrepieces. The protein solution (0.3-0.5 ~) was sedimented at 52 000 rev./min in 0.05 M Tris/chloride, 0.2 M NaCI, pH 7.4, at 22 °C. The observed sedimentation coefficient was corrected to values for a solvent with the viscosity and density of water at 20 °C (s20.w) using a partial specific volume of 0.73 [20]. The distribution of components was estimated from the area under the Schlieren peaks after correction for radial dilution. Buffers. All phosphate buffers were prepared by mixing appropriate amounts of NaH2PO4 and K2HPO4. Tris/chloride buffers were prepared by the addition of HC1 to Tris base (Sigma). Preparation of chemical variants of alkaline phosphatase. A succinyl variant of alkaline phosphatase was prepared by addition of 2 mg of succinic anhydride (Eastman) to each ml of a 0.6 ~ solution of alkaline phosphatase in 0.05 M Tris/chloride, pH 8.0 at 0 °C. The pH was maintained at 8.0 by the addition of 2 M NaOH until there was no further consumption of base. Approximately 40 70 of the lysyl residues were modified as estimated by ninhydrin analysis [21]. The nitrotyrosyl derivative of alkaline phosphatase was prepared by reaction (20 °C, 3 h) of a 60-fold molar excess of tetranitromethane (Aldrich) with 0.4~ alkaline phosphatase in 0.1 M Tris/chloride, 1 M NaC1, pH 9.0 [14]. The reaction was terminated by addition of glutathione to a final concentration of 0.03 M, followed by dialysis of the nitrotyrosyl derivative into 0.05 M Tris/chloride, pH 8.0. A total

265

®

I•RIGIN

®

!

E-NO 2 E-NO2(D/R)

F ° "3 (D/R)

E-Succ. E

Fig. 1. Hybridization of the nitrotyrosyl (E-NO2) and succinyl (E-Succ.) derivatives of alkaline phosphatase. The hybridized mixture [E-NO2 + E-Succ.] (D/R), contained 40 ~ succinyl and 60 ~ nitrotyrosyl alkaline phosphatase. Cellulose acetate electrophoresis was conducted for 15 rain at 200 V and the membrane stained for enzyme activity. of 6.4 nitrotyrosyl residues per molecule o f alkaline phosphatase were detected based o n a m o l a r extinction coefficient of 3800 for the nitrotyrosyl residue at 428 n m [22]. The a m i n o t y r o s y l derivative was prepared by reduction of the nitrotyrosyl derivative with 5 ~ (v/v) o f 0.2 M Na2S204 in 1.0 M Tris/chloride, p H 8.0.

0-5

0.4

12 i 10 ^

i0.3 8 ~

e~

0.2

o 0-1 2

20

30

40

50

60

FRACTION

70

III L ~ . 0 90 100 80

No.

Fig. 2. DEAE-Sephadex chromatography of the succinyl-nitrotyrosyl hybrid set produced by hybridization of 40 % succinyl and 60 ~ nitrotyrosyl alkaline phosphatase. The hybrid set (27 mg) was app!ied to a 1.5 cm × 30 cm column of DEAE-Sephadex A-50, pre-equilibrated in 3.10 -a M mercaptoethanol, 4" 10-a M MgCl2, 1.10 -5 M ZnC12, 0.15 M NaC1, 0.01 M Tris/chloride, pH 7.4, and eluted with a linear gradient (140 ml) of 0.15 M to 0.75 M NaC1 in the same buffer at 4°C. Fractions of 1 ml were collected and analysed for absorbance at 278 nm (e~-O) and activity in 1.0 M Tris/ chloride, pH 8.0 ( ~ - - ~ ) .

266

Hybridization experiments. The modified derivatives were hybridized by dissociation into subunits at pH 1.9 (0 °C, 1 h) and then reconstituted in 0.05 M Tris/ chloride, 1 M NaCI, 4.10 -3 M MgCI2, 3" 10 -a M mercaptoethanol, 1 • 10 -s M ZnCIa, p H 7.4 (24 °C, 20 h) resulting in a 50-70 % recovery of activity [23]. The electrophoretic pattern of the succinyl-nitrotyrosyl hybrid set shows bands corresponding not only to the nitrotyrosyl and succinyl derivatives, but an intermediate band corresponding to a hybrid with one succinyl and one nitrotyrosyl subunit (Fig. 1). The hybrid species was not detected if the derivatives were mixed without dissociation and reconstitution or if the succinyl or nitrotyrosyl derivatives were dissociated and reconstituted separately. Isolation of the hybrid species. The succinyl-nitrotyrosyl hybrid was isolated by chromatography of the hybrid set on DEAE-Sephadex (Fig. 2). Three major peaks of activity, corresponding to the nitrotyrosyl derivative (I), the hybrid species (If) and the succinyl derivative (III) can be observed. The small peak of activity (II') was found to have identical properties as the hybrid species (II). The inactive material between I and II corresponds to material that did not reconstitute during the renaturation process. RESULTS The homogeneity of the kinetic properties of the members of the succinylnitrotyrosyl hybrid set can be demonstrated by measuring the effect of sodium dithionite on the hybrid set resolved by DEAE-Sephadex chromatography (Fig. 3). The relative increase in activity (in 1.0 M Tris/chloride) on reduction of the nitrotyrosyl residues to aminotyrosyl residues (VT (+)/VT (--)) as well as the ratio of the activities of the reduced samples in 1.0 M Tris and 1.0 M NaCI (VT (+)/VH ( + ) ) are plotted versus fraction number. For both plots, plateaux in the activity ratios can be .~-o"-.-;.~ ° o ' o

°

3.G oo¢~

o

o

12

;_i

o

10

".,

>= 2.C

6~

:>

1"C

/,, 20

•

4~2

III 30

40

50

60

70

80

~

0

90

100

F R A C T I O N No.

Fig. 3. Activity ratios for the members of the hybrid set resolved by DEAE-Sephadex chromatography (Fig. 2). Enzyme activities were measured initially in 1.0 M Tris/chloride, pH 8.0 (VT(--)). An aliquot (0.1 ml) of each fraction was then reduced by the addition of 5/~1 of 0.2 M Na2S204 in 1.0 M Tris/ chloride, pH 8.0, and then assayed in 0.01 M Tris/chloride, 1.0 M NaCI, pH 8.0 (VH(+)), or 1.0 M Tris/chloride, pH 8.0 (vT (+)). The ratios of activities are given for a constant volume of each fraction.

267

ORIGIN

m

-tE -Suc¢.

............

E "NO~

[E-NOs+E Suc¢.] (D/R)

W

,.

Fig. 4. Polyacrylamide gel electrophoresis of the members of the hybrid set. Electrophoresis was conducted according to the method of Davis [24] and stained for protein according to the procedure of Fairbanks et al. [25]. The origin refers to the top of the running gel whereas the bottom of the gel is the position of the tracking dye (Bromophenol Blue). observed for peaks I, II (and II') and III, showing that the kinetic properties of each of the active members of the hybrid set are homogeneous. The activity ratios for peaks I and III correspond to the values obtained for the nitrotyrosyl and succinyl derivatives, respectively, whereas peak II and II' have intermediate and identical values for the activity ratios as might be expected for the hybrid species. The homogeneity of the members of the hybrid set is further supported by polyacrylamide gel electrophoresis (Fig. 4). Single bands are obtained for I and III with the same mobilities as the nitrotyrosyl and succinyl derivatives, respectively, whereas the hybrid (II) has an intermediate mobility. A component with low mobility can also be detected on electrophoresis of sufficient amounts of II. This species is also present in the hybrid set and corresponds to inactive material that did not reconstitute after dissociation of the variants during the hybridization procedure (see Methods). The identity of the hybrid species was further supported by dissociation and reconstitution of samples II and II'. All members of the hybrid set were produced with a distribution consistent with a hybrid containing equal amounts of succinyl and nitrotyrosyl subunits (Fig. 5). The kinetic properties of the chemical variants and the succinyl-nitrotyrosyl and succinyl-aminotyrosyl hybrids are summarized in Table I. The specific activity of the succinyl derivative is about 40 70 less than that of the native enzyme and furthermore its relative activity in Tris/chloride as compared to NaC1 (V.r/V,) is lower than the native enzyme. The modification of 6 tyrosine residues of alkaline phosphatase with tetranitromethane did not substantially affect the activity, a result reported earlier by Christen et al. [14]. However, the reduction of the nitrotyrosyl derivative with sodium dithionite to give the aminotyrosyl variant results in a large increase in

268

ORIGIN

I

(D/R)

II'

II (D/R)

II

Fig. 5. Dissociation and reconstitution of the hybrid species. The concentrated pooled fractions (II and II') were dissociated at pH 1.9 and then reconstituted at neutral pH as described in Methods. Cellulose acetate electrophoresis was conducted for 15 rain at 200 V and the electrophoretogram has been stained for enzyme activity (see Methods). e n z y m e activity. F u r t h e r m o r e , t h e r e l a t i v e a c t i v i t y o f the a m i n o t y r o s y l e n z y m e in 1.0 M T r i s / c h l o r i d e (vx) is g r e a t e r t h a n in 1.0 M N a C I (Vn) as c o m p a r e d t o t h e n a t i v e e n z y m e . I n the Tris assay, a s u b s t a n t i a l p r o p o r t i o n o f the p h o s p h a t e f r o m p - n i t r o p h e n y l p h o s p h a t e is t r a n s f e r r e d to t h e Tris buffer p r e s u m a b l y to f o r m a T r i s - p h o s p h a t e ester ( t r a n s f e r a s e activity), w h e r e a s l o w e r activities are o b t a i n e d in t h e assay in N a C 1 since t h e o n l y a c c e p t o r f o r p h o s p h a t e is w a t e r ( h y d r o l a s e activity).

TABLE I KINETIC PROPERTIES OF A L K A L I N E PHOSPHATASE VARIANTS The specific activity of the modified enzymes is given as the units of activity per mg of material that maintains the same quaternary structure as the native enzyme. Sedimentation velocity experiments showed that 21 9/ooof the nitrotyrosyl enzyme sedimented slower than the major component which had an s20,w equal to 6.5 S. In contrast, all of the suecinyl enzyme sedimented as single peak with s20.w ~ 6.5 S. Variant

Native enzyme Succinyl variant Nitrotyrosyl variant Aminotyrosyl variant" Succinyl-nitrotyrosyl hybrid ~ Succinyl-aminotyrosyl hybrid

Specific activity (units/mg) 1.0 M Tris/Cl (vr)

1.0 M NaC1 (vn)

R = vr/vri

42 23 38 114 28 65

25 15 17 37 16 25

1.7 1.5 2.2 3.1 1.8 2.6

• Based on the specific activity of the nitrotyrosyl variant and the relative increase in activity on reduction of the nitrotyrosyl residues to aminotyrosyl residues. b The specific activity of the hybrid was calculated at the position of maximum activity for peak II on DEAE-Sephadex chromatography (Fig. 2) using a specific extinction coefficient (0.1 ~ , 1 cm) at 278 nm of 0.81. This extinction coefficient is the average of the extinction coefficient at 278 nm for the succinyl and nitrotyrosyl variants.

269 The specific activities of the hybrids in the different assay mediums have intermediate values between the specific activities of their component subunits. However, it should be noted that the ratio of activity in 1.0 M Tris/chloride to that in 1.0 M NaC1 for the succinyl-aminotyrosyl hybrid is closer to that of the aminotyrosyl derivative than the succinyl derivative, indicating that the kinetic properties of the hybrid are more reflective of the more active subunit. DISCUSSION The interpretation of the kinetic properties of the alkaline phosphatase hybrids depends not only on the mechanism of the reaction and the intrinsic activities of the subunits in the hybrid but also on the degree and type of interactions between the subunits. A large body of evidence [8-10, 26-30] has accumulated that supports a reciprocating or flip-flop subunit mechanism [9]. In this mechanism, first one subunit then the other subunit must turn over in the catalytic reaction. The dimeric enzyme consequently alternates between two structures: (1) a structure in which one subunit, X, is functionally active and the other subunit, Y, is in an inactive phase and (2) the opposite structure in which subunit Y is active and subunit X is in an inactive phase. For a homogeneous dimer containing 2 identical subunits, these structures are simply mirror images and thus chemically identical. However for a hybrid species containing 2 different subunits, these structures are chemically distinct and the rate of turnover of these structures will be different. If the rate constants for the turnover of these 2 structures are given by k~ and k2, respectively (in units/rag), then the activity (v) of the hybrid will be:

v=

2 kl k2

kl-q-k2

(E~)

(1)

As v/(ET) is simply the specific activity (A) of the hybrid, we can rewrite Eqn 1 as shown below:

k2A kl-- 2k2--A

(2)

Since all the terms in this equation must be positive, each of the component subunits must turn over at a minimum rate of A/2 (i.e. k~ (and k2) ~ A/2). Consequently, the minimum turnover rate in 1.0 M Tris/chloride of the succinyl subunit in the succinylaminotyrosyl hybrid must be 33 units/mg (see Table I), whereas the rate of turnover of the succinyl variant in this assay medium is only 23 units/mg. This result shows that if a reciprocating subunit mechanism is valid for this hybrid, the interaction of the aminotyrosyl subunit with the succinyl subunit must result in activation of the succinyl subunit. The extract rate of turnover of each hybrid subunit in a reciprocating subunit mechanism can be calculated if it is assumed that the relative effect of a subunit on its partner subunit is independent of whether the assay is conducted in 1.0 M NaC1 or 1.0 M Tris/chloride. If the activity in 1.0 M NaC1 of a succinyl subunit in the

270 succinyl-aminotyrosyl hybrid is given by Eqn 2 then the activity in 1.0 M Tris/chloride of the same subunit in the same hybrid would be k~', as given below: (R2 k2) (RrtA) k',=Rlkl=zRz~z=~-~H A

(3)

where k~ and k2 are the turnover rates in 1.0 M NaC1 of the succinyl and aminotyrosyl subunits, respectively. Substitution of Eqn 2 in Eqn 3 gives the following expressions for k~ and k2: k,

:

Rn (R2 -- R,) A 2Ra(Rz--Rn) andk2=

Rn (Rz -- R 0 A

2R2(RH--R0

(4)

In the above expressions, RI, R2, and Rn are the relative activities in 1.0 M Tris/ chloride compared to 1.0 M NaC1 for the succinyl variant (RI = 1.5), the aminotyrosyl variant (R2 -- 3.1), and the hybrid (Rn ~ 2.6), respectively, and A (25 units/ mg) is the specific activity of the hybrid in 1.0 M NaC1 (See Table I). Substitution of these values in the above equations give a specific activity in 1.0 M NaC1 of 69 units/ mg for the succinyl subunit and 15 units/mg for the aminotyrosyl subunit in the succinyl-aminotyrosyl hybrid. Such a result does not seem probable as it requires the succinyl subunit in the hybrid to turn over at 4 times the rate of the succinyl variant whereas the aminotyrosyl subunit in this same hybrid would have to function at less than one half of the rate of the aminotyrosyl variant. Consequently, it would seem reasonable to conclude either that (a) the modification of the activity of a subunit by its partner subunit is dependent on the assay medium or (b) the reciprocating subunit mechanism is not valid for the succinyl-aminotyrosyl hybrid. In this regard, recent experiments by Bloch and Schlesinger [I1, 12] have indicated that the alkaline phosphatase subunits may function independently in a non-reciprocating manner. For this mechanism, we can write the specific activity for the hybrid (A) as the average of the turnover rates for each of the subunits as shown below: A = v/EO --

k , + k~

2

(5)

and thus kl=2A--k2

(6)

Since all terms are positive in the above equation, the maximum turnover rate of any subunit in the hybrid is 2A. If it is assumed, as before, that the relative effect of a subunit on the activity of its partner subunit is independent of the assay medium, then the turnover rate in 1.0 M Tris/chloride of the succinyl subunit in the succinylaminotyrosyl hybrid (kl') will be given by Eqn 7: kl' = Rx kl = 2 RHA -- Rz kz

(7)

271 where all the terms are defined as previously and Eqn 6 gives the activity of the hybrid in 1.0 M NaCI. Combining Eqns 6 and 7 gives the following expressions for k~ and k2: kl

=

2 (R2 -- Rn) A and kz = ( R 2 - - R1)

2 (RH -- R~) A

(8)

( R 2 - - R1)

Substitution of the experimental values (Table I) into the above equations shows that for an independent subunit mechanism, the succinyl subunit must turn over in 1.0 M NaC1 at a rate of 16 units/mg whereas the aminotyrosyl subunit must turn over at a rate of 35 units/mg in this hybrid. These rates are identical to the turnover rates of succinyl and aminotyrosyl subunits in the respective succinyl and aminotyrosyl derivatives and thus provides support for an independent subunit mechanism for the succinyl-aminotyrosyl hybrid. Although the experimental results for the hybrids can be explained very simply by an independent subunit mechanism it might be premature to conclude that the subunits function independently in the native enzyme. It is possible that the chemical modification of the enzyme has destroyed any negative cooperative interactions between the subunits. Although recent hybridization experiments with genetic variants [12] have given similar results with the same conclusion concerning the independent subunit mechanism, changes in cooperative interactions might possibly have occurred in the genetic variants. Only by further investigations of a variety of genetic [17, 31] and chemical hybrids under different conditions and for different steps in the enzyme mechanism will it be possible to determine if the subunits of alkaline phosphatase function independently under all conditions. Similar hybridization experiments on enzymes with proposed reciprocating subunit mechanisms, such as horse liver alcohol dehydrogenase [32] and malate dehydrogenase [33, 34] as well as other negative cooperative enzymes [35], may also provide a critical test for these mechanisms. The necessary type of variants for hybridization and the particular kinetic studies in these cases will depend on the specific type of proposed homotropic interactions. Such studies should allow the determination of whether the apparent negative cooperativity is due to an allosteric interaction [36, 37] or the presence of different chemical forms of the same enzyme. ACKNOWLEDGEMENTS We would like to express appreciation to Dr. R. E. MacKenzie, Department of Biochemistry, McGill University, for his valuable advice and suggestions during the course of this work. Financial support was received from a grant (MA-4314) from the Medical Research Council of Canada. REFERENCES 1 2 3 4 5

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