Chemistry and subunit structure of yeast hexokinase isoenzymes

ARCHIVES

OF

Chemistry

BIOCHEMISTRY

and

AND

Subunit

JAMES Department

of Microbiology,

168, 458-470 (1973)

BIOPHYSICS

Structure

J. SCHMIDT2 Vanderbilt

of Yeast SIDNEY

AND

University

Hexokinase

Isoenzymes’

P. COLOWICK3

School of Medicine,

Nashville,

Tennessee SY.23$

Received April 6, 1973 Evidence from ultracentrifugation, sodium dodecyl sulfate electrophoresis, peptide mapping, and carboxypeptidase A digestion allows the conclusion that the two native hexokinases, P-I and P-II, consist of polypeptide chains having molecular weights slightly higher than 50,000. It was demonstrated that some preparations are contaminated with a protease, and that this impurity caused erroneous results in sodium dodecyl sulfate electrophoresis and carboxypeptidase A digestion. Amino acid analyses indicated that both P-I and P-II contain four cysteine, four tryptophan, and eleven methionine residues per mole. In contrast, P-I contains eight, and P-II five, histidine residues per mole. Many of the differences in amino acid Composition are small, but reproducible. Peptide mapping indicated that many segments of P-I and P-II have identical sequences. There were about 27 common tryptic peptides, and about 16-19 unique to each form. In addition, both isozymes were found to have the same amino terminus, valine, and the same carboxy terminus, alanine; some evidence for a difference in the penultimate residue at the carboxy terminus was indicated.

Hexokinase (ATP : n-hexose 6-phosphotransferase, E.C. 2.7.1.1.) purified from baker’s yeast according to earlier procedures (1, 2), was found to contain multiple chromatographic forms (3, 4). Subsequently, it was discovered that the preparations contained proteases (5-7), and that this impurity could lead to the production of altered forms of the enzyme (6, 7). Methods were developed to remove and/or inactivate these proteases, and allowed the isolation of hexokinase in two distinct forms, designated P-I and P-II (6-9). Similar results have been reported from another laboratory (10). P-I and P-II are separate, noninterconvertible isozymes, and are thought to be the 1 Supported by USPHS Grant No. AM 03914 and USPHS Predoctoral Fellowship No. 2-F lGM-36,400. This manuscript is based on a portion of the dissertation submitted by Dr. J. J. Schmidt for the Ph.D. degree, Vanderbilt University, May 1971. 2 Present address: Division of Biological Sciences, Indiana University, Bloomington, Indiana 47401. 3 To whom correspondence should be addressed.

native forms which occur naturally in the yeast cell (6, 7). They may be differentiated b;v several criteria (7-9). Under many conditions, for example, at low ionic strength and neutral pH, the native P-forms have molecular weights of about 100,000 (11-16). Dissociation to a 50,000 MW form can occur (7, 12-16); furthermore, in denaturing solvents, a 20,000-26,000 MW species has been reported (12, 17-19). Recently, however, an ultimate subunit MW of 50,000 was reported from this laboratory (l5), and this finding is documented in the present report. It was previously found (6) that very low concentrations of trypsin (trypsin to hexokinase ratio: 1 to 10,000) could convert P-I and P-II to new, fully active isozymes, designated S-I and S-II, respectively. The conversion requires that the P-form be dissociated, by means of high ionic strength or glucose addition, to its 50,000 MW species. The S-forms tend to exist mainly as the 50,000 MW forms, and associate to higher molecular weight oligomers only at pHs near their isoelectric points (6, 7). The catalytic properties of the P-forms are re-

458 Copyright All rinhts

@ 1973 by Academic Press, of rmroductioo in anv form

Inc. reserved.

CHEMISTRY

OF YEAST

HEXOKINASE

ISOENZYMES

459

Other chemicals and their sources were: 5-5’tained, although a small increase in specific dithio-bis-2-nitrobenzoic acid (DTNB)4, Aldrich activity of S-I over P-I has been observed Chemical Company; dithiothreitol, Calbiochem; under certain conditions. methyl mercuric iodide, K and K Laboratories, These catalytic assays were carried out at Inc.; Coomassie blue and sodium dodecyl sulfate very low protein concentrations, where both (SDS), Sigma Chemical Company; 30$& hydrogen P and S forms are presumed to be in the dis- peroxide, Merck and Company, Inc.; and ninsociated form. At high protein concentrahydrin in aerosol cans, Nutritional Biochemirals tions, where the S-forms are dissociated but Company. the P-forms are not, the S-forms have been Worthington Biochemical Corporation was the shown to bind glucose more tightly than the source of the following proteins: L-(l-tosylaminoketone -treated P-forms (20), indicating that the active site 2-phenyl) -ethyl-chloromet,hyl becomes less accessible upon association of trypsin, carboxypeptidase A (diisopropyl fluorophosphate-treated), hen egg white lysozyme, and the subunits. soybean trypsin inhibitor. Since the abovementioned findings indiBovine serum albumin (BSA), pyruvate kinase, cated that the transformation of P to S lactate dehydrogenase, triose phosphate deh)-forms had profound effects on subunit interdrogenase, gamma globulin, ovalbumin, aldolase, action and substrate-binding properties, a and glutamic dehydrogenase were all purchased detailed study of the chemistry of this from Sigma Chemical Company. Hemoglobin was transformation \yas undertaken with both a product of Nutritional Biochemicals Company. native forms P-I and P-II. This study rePurification and ilssay of Yeast Hexokinase quired, as prerequisites, more information The purification method was that, of Schulze on the chemistry and subunit structure of et ~2. (8) with the modifications described in the the native enzymes. preceding paper (9). Hexokinase activity was In the present report, data are presented measured with the cresol red assay of Darrow and on the following: (1) A further comparison Colowick (2). One unit of hexokinase catalyzes of the hexokinases with respect to amino acid the phosphorylation of 1 pmole of glucose per content and end group analysis. (2) An minute at 30°C and pH 8.9. investigation of the subunit structures of The concentration of the pure hexokinases in both P and S forms. (3) The discovery of mg per ml can be estimated by multiplying the erroneously low molecular weights in sodium optical densit,y of the solution at 280 nm by 1.1 (21, 22). When actual prot.ein concentration was dodecyl sulfate solution due to contamination of some hexokinase preparations by a determined by amino acid analysis (see belowj. factors of 1.13 and 1.06 were found for P-I and proteolytic enzyme. MATERIALS

AND

METHODS

Chemicals All chemicals were reagent grade, except where otherwise noted. Guanidine hydrochloride (Eastman Organic Chemicals) and urea (J. T. Baker Chemical Co.) were recrystallized from 95% ethanol and stored at 4°C. In some cases, Ultrapure Grade guanidine HCI and urea from Schwarz/Mann were used without further purification. The following chemicals were obtained from Eastman Organic Chemicals: 2-mercaptoethanol, acrylamide, N,X’-methylene-bis-acrylamide, and tetra methyl ethylene diamine. Butanol, pyridine, and formic acid were obtained from Fisher Scientific Company. Sodium iodoacetate was obtained from Sigma Chemical Company. The product received was faintly yellow. However, recrystallization from 95y0 ethanol eliminated the color.

P-II, respectively. These correspond to values of 8.85 and 9.47, respectively, for A:%,. Previously, a value of 9.16 was reported for P-II (16). The Lowry (23) and biuret (24) methods were also done using BSA as standard. Since all three methods gave virtually the same results with a given hexokinase solution, the spectrophotometric method was often employed. Where the concerttration of hexokinase is expressed in moles, it is assumed that one mole of the enzyme rorresgonds to approximately 50,000 g.

Preparation of S-Forms The native hexokinases were converted to the corresponding S-forms essentially by the method of Schulze and Colowiek (6, 7) with modifications described in the following paper (25). 4 Abbreviations used: BSA, bovine serunl alCX, carboxymethyl-; DTNB, j-5’bumin; dithio-bis-2-nitro-benzoic acid; SDS, sodium dodecyl sulfate.

460

SCHMIDT

AND COLOWICK

SDS Electrophoresis This technique was carried out according to the method of Weber and Osborn (26). Modifications of gel and buffer compositions are described in the Results section. In most cases, proteins were reduced and carboxymethylated according to the procedure described below, then dialyzed against the appropriate electrophoresis buffer containing SDS. Samples which were not carboxymethylated were first treated with 0.1 M dithiothreitol and SDS. Bromphenol blue and 0.3 M sucrose were included in each sample. After electrophoresis, gels were removed from the tube, sliced with a razor blade at the location of the tracking dye band, and stained according to the method of Weber and Osborn (26). Relative mobility, then, equals the distance migrated by the protein divided by the length of the gel. Molecular weights of the marker proteins were obtained from Weber and Osborn (26), with the exception of soybean trypsin inhibitor (27).

Chemical ModiJication

of the Hexokinases

The method of reduction and carboxymethylation was essentially that of Crestfield et al. (28), while performic acid oxidation was done according to the procedure of Moore (29). In some cases, the sulfhydryl groups of hexokinase were blocked by reaction with methylmercuric iodide (30).

Amino Acid Analyses The amino acid content of the hexokinases was determined by the method of Moore and Stein (31). The analyses were done either with a Beckman Model 120C Amino Acid Analyzer or with a Technicon Autoanalyzer. The Beckman instrument was equipped with an Infotronics Model CRS-12-AB integrator and a 45.1 MV resistor (“range”) card.

Tryptophan

Content of the Hexokinases

Tryptophan was measured by three different methods (32-34) as described in the Results section.

Carboxypeptidase A Solutions of carboxypeptidase A were prepared essentially according to the method of Potts (35). A hexokinase to carboxypeptidase A ratio of 5O:l was employed during digestion, and results were quantitated on a Beckman Model 120C Amino Acid Analyzer.

Peptide Mapping Carboxymethylated hexokinases were equili-- - brated with 0.4 M ammonium bicarbonate, pH 8.0,

at a concentration of 10 mg of protein per milliliter and digested for 8 hr at room temperature with trypsin. The initial concentration of trypsin was 1% of the hexokinase concentration (by weight); after 4 hr, fresh trypsin was added to give a final concentration of 20/, of the hexokinase concentration. In some cases, the digestion was done in a Radiometer TTT-lb Automatic Titrator. Trypsin, dissolved in 1 mM HCl, was added to give a concentration of 2% of the hexokinase concentration, and the pH was maintained at 8.0 by the addition of 0.05 M NaOH (standardized against potassium hydrogen phthalate). The reaction was allowed to proceed at room temperature under argon until the rate of addition of NaOH returned to the baseline rate, usually in 4-5 hr. Peptide maps were then prepared basically as described by Bennett (36). The lyophilized digests (representing 20-40 nmoles of protein) were dissolved in pH 6.5 10% pyridine-acetic acid and spotted on 46 X 57 cm sheets of Whatman 3MM chromatography paper. Electrophoresis was carried out in the abovementioned solvent for 3 hr at 35 V per centimeter. The papers were then completely dried, and chromatographed in the second dimension in butanol/acetic acid/water/ pyridine (15/3/12/10) for 18 hr. Peptides were detected with a ninhydrin spray, followed by the Pauly spray for the visualization of histidinecontaining peptides (37, 38).

Ultracentrifugation The samples were loaded into 12 mm double sector ‘cells on top of a small amount of FC-43 (perfluorotributylamine, Minnesota Mining and Manufacturing Company). The length of the liquid columns in the cells was about 3 mm. A sixhole rotor was used, so that five different protein concentrations could be run at once; ordinarily, these concentrations ranged from 0.1 to 3.0 mg per ml. In some experiments, high speed sedimentation equilibrium runs were done according to the method of Yphantis (39) in a Spinco Model E Ultracentrifuge equipped with a Rayleigh interference optical system. The apparent molecular weight was evaluated with Svedberg’s equation: Mjjr..-

2RT d In c , (1 - BJo dr=

where the symbols have their usual meaning. The value for the partial specific volume of hexokinase used in the equations was 0.735 ml/g calculated from the amino acid composition (46). The photographic plates from the ultracentrifuge were analyzed with a Nikon Model 6

OF YEAST

CHEMISTRY

HEXOKINASE

Microcomparator and the data calculated with the aid of programs written for a Wang 360 Calculator. The molecular weight in a given solvent was then calculated from plots of apparent molecular weight versus protein concentration, extrapolated to infinite dilution. In other experiment,s, a Spinco Model E Ultracentrifuge fitted with a photoelectric scanner and multiplex accessory unit was employed. The samples were centrifuged at 13,000 rpm until equilibrium was attained, then the cells were scanned at 280 nm and plots of optical density versus distance obtained. A built-in calibration mode and a reference cell allowed direct calculation of In C and r2 from the chart recorder paper. A small correction for baseline absorption was made by accelerating the rotor to 40,000 rpm, thereby deplet,ing the meniscus of prot,ein. Again, the apparent molecular weight was calculated from Svedberg’s equation, and the final value obtained from a plot of apparent molecular weight versus prot,ein concentration. The method is described in more detail by Chervenka (41). RESULTS

Amino Acid Compositions of the Hexokinases 1. Amino acid recoveries following acid hydrolysis. Samples of P-I and P-II mere

461

ISOENZYMES

hydrolyzed in 6 N HCl for various intervals, and the results for various intervals are presented in Tables I and II. For P-I, the calculations were based on the assumption t’hat 38 molrs of lysine are present per mole of protein. Thr recoveries of amino acids at various time intervals are good, except for tprosine, which is partially destroyed. In several other 24-48 hr hydrolyses, 14.5-15 molts of tyrosirw per mole were routinely obtained. Also, if a plot, of recovery versus time is made using t*he data in Table II, 14.9 residues per molr are swn at zero time. Thercforc, t,yrosirw has been assigned a value of 15 residues pc’r mole. The results for I’-11 ww calculated by assigning a value of 34.0 rcsiducs pw mole to lysine. In this case, tyrosiw n-as not destroyed, so no correction was applictd. These samples were carl)oxvmeth~latt~d; notth that four carboxymethyl cystcmr rcwiduw prr mole are rwowwd. Lysine was chosen as a basis for calculation because w&h many hydrolysw under a variety of circumstances, little doxtruction of t’his amino arid n-as w(ar ww. Residues

TABLE I AMINO ACID COMPOSITION OF P-I Residues per mole

Residue Hydrolysis

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

time (hours)

23

47

71

95

38.0 8.3 17.9 57.0 29.3 22.3 52.1 26.8 41.8 31.7 26.1 10.8 28.1 47.8 14.2 17.9

38.0 8.3 18.1 55.7 28.7 21.8 51.3 24.9 41.8 32.1 24.7 10.6 28.3 48.5 13.2 17.4

38.0 8.5 17.6 56.5 28.3 20.6 51.9 24.9 42.2 33.0 27.7 10.7 30.3 48.8 12.4 17.5

38.0 8.2 17.G 56.1 27.4 19.6 51.7 24.7 41.9 32.8 28.3 10.4 30.1 48.5 11.7 17.5

Extrapolated or average valuen 38.0 8.3 17.8 56 .:3 29.9 23.3 51.7 25.3 -12.2 32.4 26.i 10.6 30.1 48.5 15.0h 17.G

Nearest integer

38 8 18 56 30 23 52 25 ‘42 32 27 I1 30 49 15 18

Q Threonine and serine are zero-time values obtained by extrapolation; valine, isoleucine, and leucine are the maximum amounts recovered. b In this experiment, tyrosine was partially destroyed. The value of 15 was obtained from a plot of recovery versus time.

462

SCHMIDT

AND TABLE

COLOWICK II

AMINO ACID COMPOSITION OF CM-P-II Residue

Residues per mole Hydrolysis

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isolucine Leucine Tyrosine Phenylalanine CM-cystine a Values for threonine leucine are the maximum

48

72

96

Extrapolated or average valuea

Nearest integer

34.0 4.5 17.8 53.1 28.2 24.4 54.9 29.6 39.8 32.6 20.4 10.8 34.0 35.1 15.1 22.7 3.7

34.0 4.7 18.2 53.7 27.9 23.5 54.4 28.3 39.5 32.7 22.1 11.0 35.3 34.7 15.5 23.1 4.1

34.0 4.7 17.9 53.1 27.1 22.4 54.5 28.5 39.7 32.7 23.7 11.2 36.0 34.9 15.1 23.0 3.9

34.0 4.7 17.8 52.1 26.8 21.9 53.7 28.0 39.4 32.3 24.1 10.9 35.9 33.7 15.0 23.7 3.7

34.0 4.7 17.9 53.0 28.9 25.2 54.4 28.6 39.6 32.6 24.1 11.0 35.9 34.9 15.2 23.1 3.8

34 5 18 53 29 25 54 29 40 33 24 11 36 35 15 23 4

and serine are extrapolated amounts recovered.

present in small numbers such as histidine, methionine, tyrosine, and cysteine are not always sufficiently stable to justify their use as calculation standards. Physical studies indicate that the molecular weights of P-I and P-II are somewhat higher than 50,000; those particular values of lysine were chosen because they produced amino acid compositions corresponding to molecular weights slightly larger than 50,000. However, in the absence of sequence data for the entire monomer, no exact molecular weight can be obtained; hence, an approximate value of 50,000 is assumed for both P-I and P-II in this report. 2. Cysteine content of the hexokinases. It has been reported (6) that P-I contains four, and P-II contains three sulfhydryl groups per 50,000 MW, and no disulfide bonds. However, the results reported herein (Tables II and III) indicate that both forms contain four sulfhydryl groups per mole. This is based on carboxymethylation (28), performic acid oxidation (29), and reaction with DTNB (42). 3. Estimation

time (hours)

24

of the tryptophan

content of

to zero time.

Values

for valine,

leucine,

and iso-

When the tryptophan content of the hexokinases was estimated spectrophotometrically by the method of Goodwin and Morton (32), it was found that P-I contained 3.4, and P-II 3.5 tryptophans per 50,000 MW. This was based on the presence of 15 tyrosines per 50,000 MW for both P-I and P-II. A similar procedure, using guanidine hydrochloride as the solvent (33), was attempted. This method gave 4.1 and 3.9 tryptophans per 50,000 MW for P-I and P-II, respectively. Finally, the procedure of Matsubara and Sasaki (34), amino acid analysis following hydrolysis in 6 N HCl-thioglycollic acid, was tried. Results were calculated using a value of 18 arginines per mole for both forms. On this basis, 3.4 tryptophans per mole were recovered from P-I, and 3.1 tryptophans per mole from P-II. Since yields in this procedure were generally 80-90 % (34), corrected values in the range 3.84.2 and 3.4-3.9 may be assumed. With all three methods, different hexokinase preparations gave quite similar reP-I and P-II.

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HEXOKINASE

463

ISOENZYMES

Note that for both P-I and P-II, alanine is the carboxy terminal residue, confirming earlier results of Gazith et al. (6). Also important is the fact that approximately one Carboxypeptidase A Digestion residue of alanine per 50,000 MW is obtained Carboxymethylated P-I and P-II were at 12 hr with both P-I and P-II, suggesting digested with 2 % of their weight of carboxy- the presence of a single polypeptide chain of peptidase A and aliquots were removed and that size. This is in contrast 60 t,he earlier analyzed at the times indicated in Tables studies (6), in which were found two alanines IV and V. After removal of 12 hr samples, per 50,000 MW. With P-I, the amount of alanine does not fresh carboxypeptidase A was added to a exceed one residue per 50,000 MW even final concentration of 3 70 of the weight of after 24.5 hr of incubation. The only other hexokinase present. amino acids seen are very small amounts of lysine, isoleucine, and lcucine. TABLE III

sults. Thus, one may conclude that both P-I and P-II contain three to four, probably four, tryptophan residues per 50,000 MW.

CE~STEINE

CONTENT

Measurement Carboxymethyl cysteinea Cysteic acid* Reaction with I)TNBc

OF

P-I

AND

P-II

Peptide Mapping

Residues per mole P-I P-II 3.7, 3.8

3.9, 3.6

4.1, 3.9 3.9, 3.7, 3.6

4.0, 4.0 3.9, 3.6, 3.8

= Amino acid analysis after acid hydrolysis of carboxymethyl derivatives of enzyme. Calculations based on comparison with lysine recovery (see text). Values for two different preparations of each enzyme are shown. * Amino acid analysis after performic acid oxidation. Calculations based on comparison with aspartic acid recovery. Values for two different preparations of each enzyme are shown. c Moles of sulfhydryl group per mole of enzyme. The three values shown for each enzyme are for the following conditions, respectively: (1) 8.7 M urea, 0.05 M potassium phosphate, pH 8.0; (2) 6 M GuHCl, 0.05 M potassium phosphate, pH 8.0; (3) 0.2 M ammonium bicarbonate, pH 8.2. In the latter case the measurements were made after 3 hr of incubation with DTNB at room temperature. In the first two cases, reaction was virtually instant,aneous.

AMINO

ACIDS

RELEASED

TABLE IV CM-P-I

FROM

Residue

Alanine Isoleucine Leucine Lysine

The technique of pf>ptide mapping was applied for two reasons: (1) to compare further the structures of the hcxokinase isozymes, and (2) to obtain information on the subunit molecular ncight of each isozyme. Typical patterns are shown in Figs. 1 and 2. The blackened spots are common to both P-I and P-II, while the unshaded ones are unique t’o each form. These were determined by comparing many individual maps, including some which were mixtures of P-I and P-II on the same paper. From these data, one may conclude that there are about 27 common and 16-19 unique tr!pt.ic peptides. In Fig. 2, two peptidcs in the upper lefthand corner are indicated by arrows. These are lost when a P-form is converted to an S-form. They are common peptides, and the lower one cont’ains histidine (detected by Pauly spray). It should be noted that the maps acre very reproducible with different batches of t’he hexokinases, regardless of whet,her BY CARBOXYPEPTIDASE

A

Moles of amino acid per 50,000 MW 2 hr

5 hr

8 hr

12 hr

0.54 Trace Trace 0

0.77 Trace Trace 0

0.85 Trace Trace 0

0.83 0.05 0.04 0

24.5 hr 0.90 0.07 0.04 0.04

464

SCHMIDT

AND COLOWICK

peptide chain of about 50,000 MW or, if there are smaller subunits, they are not identical.

digestion was done in ammonium bicarbonate buffer or in the Radiometer pH-Stat. In the latter case, calculations indicated cleavage of approximately 50-60 bonds per 50,000 MW, suggesting complete digestion of the enzyme by trypsin. A consideration of the peptide maps can provide information on the subunit structure of the molecules. Considering the amino acid analyses of the isozymes, the presence of two identical subunits of approximately 25,000 MW would result in less than 30 peptides. However, since 4046 peptides were routinely observed with both P-I and P-II, there must be eit,her a single polyTABLE

Alanine Lysine Leucine Isoleucine Serine Glycine Histidine Valine Threonine

1. SDS-electrophoresis. Figure 3 shows a plot of molecular weight versus relative migration for a set of standard proteins and for P-I and P-II. Note that many of the standard proteins are subunit enzymes. In this experiment, both hexokinases were found to have a molecular weight of 51,000. The technique was repeated many times under various conditions, with S-forms as well as P-forms. In all cases,values approximating 50,000 were obtained for the molecular weights of the hexokinases. Occasionally, faint bands of lower and higher molecular weights were seen, but never at the location corresponding to 25,000 MW. Table VI summarizes the conditions and results for each experiment. 2. Ultracentrifugation. Figure 4 shows a plot of apparent molecular weight versus protein concentration for two separate runs. The lower plot depicts the results obtained for P-II treated with methyl mercuric iodide, dissolved in 6 M guanidine HC1-0.2 M Trisglycine, pH 8.8. Except for the last few points, the values range from 51,000 to

V

AMINO ACIDS RELEASED FROM CM-P-II CARBOXYPEPTIDASE A

Residue

Ultimate Subunit Molecular Weight of Hexokinase in Denaturing Solvents

BY

Moles of amino acid per 50,000 MW 2.5 hr

6 hr

10 hr

25.5 hr

0.79 0.14 0.07 0.04 0.03 a 0 0 c.03

1.04 0.16 0.07 0.05 0.07 0.03 a.03 0 0.03

1.08 0.20 0.13 a.11 0.10 0.05 0.06 0.02 0.04

1.41 0.56 0.52 0.50 0.16 0.14 0.10 0.08 0.05

P-I

1

8 FIG. 1. Peptide map of CM-P-I.

CHEMISTRY

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HEXOKINASE

ISOENZYMES

465

Origin a

(-)

FIG. 2. Peptide map of CM-P-II.

56,000. Using the method of least squares, the extrapolat.ed molecular weight is 51,000 f 630 (excluding the first three points). The apparent increase in molecular weight at, very low protein concentrations represents a phenomenon which is as yet unexplained. For purposes of comparison, the upper plot represents native p-11 in 0.05 NI potassium phosphate, pH 7.0. Here, t’he extrapolated molecular weight is 102,700 f 5,240, exclusive of the first point (lowest concentration). The apparent decrease in molecular weight at the lowest concentration is probably due to dissociation to the 50,000 MW monomer. Under the same conditions, S-II yields an extrapolated molecular weight of 52,000. It should be noted that under both nat’ive and denaturing conditions, plots of In c versus r2 show no inflection points or deviations from linearity. That is, the molecular weights appear homogenous over the entire lengths of the solution columns. In other experiments, CM-P-II and CM-SII were ultracentrifuged in denaturants. CM-P-II was dissolved in 9 M urea-O.1 M Tris-HCI, pH 8.1, and gave an extrapolated molecular weight of approximately 52,000. CM-S-II in 6 M guanidine HClLO.1 nr borate, pH 8.9, produced a value of about 45,000. Ko suggestion of a 25,000 MW species was ever seen. Thus, one may conclude that the ultimate subunit molecular weight of hexo-

kinase in denaturing solvents is close to 50,000. 3. Erroneous molecular weights due to contamination with, proteases. At the time that these molecular weight determinations were in progress, workers in another laboratory were reporting that the subunit molecular weight of yeast hexokinase was not 50,000, but 26,000 (18). One of the methods used to obtain this result was SDS-electrophoresis, but the sample preparation was somewhat different from that described in the Methods section of the present paper. The method used by the other group was to dissolve the samples in 1% SDS-O.06 M Tris-glycine, pH 8.6, incubate briefly at 37°C and dialyze overnight against 1% SDS-O.06 Y Trisglycine, pH 8.6. Several preparations of hexokinase were obtained from the other laboratory and subjected, along wit’h several of our hexokinases, to SDS-electrophoresis following their pretreatment procedure. As usual, all the hexokinases showed a molecular weight of 50,000, except for two of their preparations, which had molecular weigh& of about 16,000. Apparently, degradation of the proteins in these two samples had occurred. However, a molecular weight of 50,000 could be obtained with these samples if they were first carboxymethylated in 6 ;II guanidine HCl. More evidence for degradation of cert.ain hexokinase samples by proteases was ob-

466

SCHMIDT

AND COLOWICK

a t

cwlbumin

t d

aldolase

Lactic Cehydrogenase

43 x

II

I I I 0.1 0.2 03

1 I CH a5

I I 0.6 07

I 08

Relattve Migrutiin

I D

FIQ. 3. SDS-electrophoresis. Both samples and standards were carboxymethylated. Other conditions: 10% acrylamide, 0.04 M Tris buffer, pH 8.0, 0.1% SDS.

tained when digestion was carried out with 10 % carboxypeptidase A in the presence of 1% SDS. This was done using one of our preparations and one of theirs which gave 16,000 MW in SDS-electrophoresis. After incubation for 3 hr at room temperature, the samples were lyophilized, dissolved in buffer, and analyzed. The results are summarized in Table VII. Note that with P-I more or less the same pattern is seen as that without SDS, although the increased amounts of serine, glycine, histidine, isoleucine, and leucine may indicate a small contamination with proteases. However, in the other preparation, a rapid release of many amino acids is observed, suggesting degradation of the molecule by a contaminating endopeptidase. DISCUSSION

Evidence has been presented in this report that the monomeric subunit molecular weight of yeast hexokinase is approximately 50,000

rather than the values of 20,00&26,006 which have been previously reported (12, 17-19). This conclusion is based on several criteria, discussed individually below. First of all, electrophoresis in the presence of SDS (26) consistently produced molecular weights near 50,090 for all four hexokinase isozymes. Usually, these were somewhat higher than 50,000 (Table VI). The method was repeated many times under a variety of conditions, but nothing was found to indicate a 25,000 MW subunit. Secondly, ultracentrifugation under dissociating conditions supported the data described above. An extrapolated molecular weight of about 51,000 was found when P-II was treated with methyl mercuric iodide and centrifuged with 6 M GuHCl included in the solvent. Similar results were obtained for CM-P-II in 9 M urea (52,000) and CM-S-II in 6 M guanidine HCl (45,000). In contrast, native P-II in 0.05 M potassium phosphate, pH 7.0, had a molecular weight of about 103,000, while that for S-II in the same solvent was 52,000. Thus the native enzyme P-II must be considered as a dimer and the modified enzyme S-II as a monomer, instead of as tetramer and dimer, respectively, as previously believed (7, 12, 17-19). In calculating the ultraeentrifugation data, it was assumed that the partial specific volumes of the hexokinases were the same both in solutions of dilute salt and in concentrated denaturants. This has been shown to be valid for several proteins (44). On the other hand, some variation has been shown to occur (45, 46). For example, Thomas and Edelstein (45) found that the apparent partial specific volume of DNA polymerase varied from 0.72 cc/g in 3 M guanidine HCl to 0.76 cc/g in 7 M guanidine HCl; that for the native protein is 0.74 cc/g. Differences were also observed for rabbit gamma globulin in dilute buffer (0.74 cc/g) and in 6 M guanidine HCl (0.72 cc/g) (46). Consequently, the accuracy of the molecular weights found for hexokinase in guanidine HCl or urea may be questioned. Nonetheless, if the partial specific volume of hexokinase varies within the ranges described above, corrected molecular weight values would not be changed sufficiently to indicate a 25,000 MW monomer. Quantitation of the products resulting

CHEMISTRY

OF YEAST

HEXOKINASE

from carboxypeptidase A digestion of CMP-I and CM-P-II also suggested a single polypeptide chain per 50,000 MW. The earlier finding (6), that alanine is carboxyterminal in both P-I and P-II, is confirmed. However, only one alanine per 50,000 NW was obtained for P-I; on prolonged digestion of P-II, part of a second residue of alanine appears, but t,his could be explained by t,hc presence of another alanine in t,he wquenw close to the end of the chain. Finally, the technique of peptide mapping implied the absence of identical 25,000 XtW subunits. The observation of 40-46 tryptic peptides supports the conclusion that hexokinase consists of approximately 50,000 &JW polypeptide chains, or, if there arc smaller subunits, they are not identical. Thus it is seen that the results from SDS elect.rophoresis, ultracentrifugation, carboxypeptidasc A digestion, and pept)ide mapping all indicate that the subunit molecular weight of hexokinase is not 25,000 but 50,000. Clearly, an cxplanabion is nwdcd for the erroneously low values report,ed previously. One factor which could account, for man)

100 A A I---.

I

I

I

I

1 2 Protein concentration

3 (mg/mll

FIG. 4. Apparent molecular weight versus protein concentration. (A---A) P-II in 0.05 M potassium phosphate, pH 7.0. (O---O) P-II + CHsHgI in 6 M (;uHC1/0.2 M Tris-glycine, pH 8.8. TABLE MOLECULAR WEIGHTS OF THE HEXOKINASES Expt no.

VI DETKRMINKD

Acrylamide concentration c%‘L)*

Buffer pH

pH 8.0

467

ISOENZYMES

BY SDS-ELECTROPHORIXSG SDS concentration (%)

1

064 M Tris-HCl,

10

0.1

2C

0.05 M Na-phosphate,

pH 7.0

9

0.1

3” 4

0.02 M Na-phosphate, Tris-glycine, pH 8.8

pH 7.0

9 9

0.1 0.1

5

Tris-glycine,

pH 8.8

9

0.1

6 7

Tris-glyeine, Tris-glycine,

pH 8.8 pH 8.8

12.5 11.25

0.1 0.5

8

Tris-glycine,

pH 8.9

10

1.0

LLExcept as not,ed, both unknowns and standards were carboxymethylated. ‘) N,iV’-methylene bis acrylamide concentration = 1/30th the acrylamide c Samples not, carboxymethylated.

Molecular weights found

P-I P-II S-I P-II S-I S-II P-I P-I P-11 S-I S-II S-I P-I P-II S-I S-II P-I S-I S-II

concentration.

50,000 51,000 52,000 53,000 47,000 50,000 53,000 53,000 52,000 54,000 58,000 48,000 50,000 50,000 49,000 54,000 50,000 48,000 51,000

468

SCHMIDT

TABLE VII AMINO ACIDS RELEASED BY CARBOXYPEPTIDASE IN THE PRESENCE OF SDS Residue

AND COLOWICK

A

Residues per mole Hexokinase Hexokinase P-I A”

Lysine Histidine Threonine Serine Glutamic

acid

Glycine Alanine Valine Isoleucine Leucine Methionine Tyrosine Phenylalanine

0.08 0.29 0.17 0.47

0.30 0.10 3.1 3.6

0.05

0.18

0.18 0.81 0.12 0.23 0.18 0 0 0

0.45 2.7 4.2 2.3 3.8 3.1 1.9 5.7

4 A sample from Barnard’s laboratory which showed a low molecular weight (ca. 16,000) by SDS gel electrophoresis.

of the low values is the possibility that most of the older preparations were contaminated with proteases. For example, Kenkare and Colowick (12), using a hexokinase preparation in which no precautions had been taken to avoid proteases, reported molecular weight values in the vicinity of 25,ooO at pH 11.5 or 2.8 by ultracentrifugation and at pH 10.5 by light scattering. However, Schulze and Colowick (7), using light scattering at pH 11.0 and preparations of enzyme presumably free of protease, found a molecular weight of 50,000. It is possible that at the extremes of pH employed by Kenkare, the denatured hexokinases were digested by proteases which remained active under those conditions. A similar view may be advanced to explain the low molecular weight observed by Ramel et al. (17) in sedimentation rate experiments in the presence of SDS. Furthermore, it has recently been shown (47) that treatment of proteins with SDS does not produce a random coil conformation, but causes them to assume a highly ordered, rodlike structure whose length is a function of molecular weight. In view of this, it is obvious that calculations of molecular weights from sedimentation velocity coefficients ob-

tained in the presence of SDS could produce highly inaccurate results. Other evidence which had been used to support the 25,000 MW subunit proposa1 includes the observation of 24-30 tryptic peptides by Gazith et al. in this laboratory (6). However, digestion was done using samples which had been denatured by boiling rather than by S-carboxymethylation, the procedure used in the present work. A certain amount of material always remained at the origin of the peptide maps with enzyme digested by the former procedure, but not with the new procedure. Also, in the earlier studies, electrophoresis was carried out at pH 3.5, in which case nearly all the peptides move toward the cathode and are not as well resolved as at pH 6.5. Another instance of apparent support for a subunit molecular weight of 25,000 was the report that carboxypeptidase A released two to three alanines per mole of protein (6), but this was after a long incubation and again, degradation by a protease could have been a factor. The contamination of some hexokinase preparations by a highly stable protease activity is strongly indicated by the results presented in two sections of this report. For example, prolonged pretreatment with SDS caused some hexokinase samples to give unusually low molecular weights on SDS electrophoresis, unless these samples were first carboxymethylated in guanidine HCI. Furthermore, one of these samples, upon treatment with carboxypeptidase A and SDS, showed a very rapid release of many different amino acids. Both observations suggest that degradation of the molecule had occurred, presumably by the action of a protease which remains active in SDS but is inactivated by the carboxymethylation procedure. Supportive evidence for this view has recently been published by Pringle (48), who found that commercial preparations of yeast hexokinase contain a protease which caused erroneously low molecular weights for the hexokmase on SDS electrophoresis. If steps were taken to inactivate this protease (e.g., heating to 100°C or dissolving the protein

CHEMISTRY

OF YEAST

HEXOKINASE

in hot 5.5 M guanidine HCl) a molecular weight of 51,000 was found. It should be emphasized that traces of proteases which are unable to cause the P to S conversion during isolation of the enzyme are nevertheless capabIe of degrading the enzyme in the presence of SDS. It appears that the denatured hexokinase molecule is much more susceptible than the native molecule to this degradation process. The problem of contaminat’ion of supposedly pure enzyme preparations by small amount’s of proteases may be more widespread than is now suspected. For example, Cassman and Schachman (49) have recently found proteolytic activity in commercial preparations of glutamic dehydrogenase. This protcnsc was a&iv? in 2-5 M guanidine HCl, but could be inactivated by higher concentrations or by 2-mercaptoethanol. If steps were not t)akrn to eliminate it, inaccurately low molecular weights resulted. In the present work, the problem of protease cont,amination was completely overcome by means of reduction and carboxymethylation in the presence of either 6 M guanidine HCI or 8 nl urea. In another main area of interest, a comparison of the two nat’ive hexokinases was undertaken. Wit,h respect to the complete amino acid composition, values of lysine were selected as calculation standards such that the resultant molecular weights were somewhat higher than 50,000, in order to agree with physical measurements. The results for the two P-forms show many similarities; for example, carboxymethylation, performic acid oxidation, and reaction with DTNB show the presence of four sulfhydryl groups per mole in both P-I and P-II. This is in contrast to the earlier report of four sulfhydryl groups in P-I, and three in P-II (6). Also, alanine is the carboxy-terminal residue in both, but a difference in the penultimate residue is indicated. A manual Edman degradation showed that valine is the amino-terminal residue of both P-I and P-II. On the other hand, P-I contains eight histidines per mole, while P-II contains five. While P-II has about the same number of

ISOENZYMES

469

isoleucines and leucines, 36 and 35, respcctively, P-I contains 30 isoleucines and 49 leucines. There is less lysine in P-II t’han P-I (34 versus 38 residues) but more glutamic acid (54 versus 52 residues). Many of the differences are smal1 but all arc quit,c reproducible. When peptide maps were prepared, it was found t’hat P-I and P-II shared about 27 common tryptic peptides, but about 16-19 were unique to each form. This indicates a high degree of similarity in sequences between the two isozymes. Nonetheless, the conformations of the native molecules are different enough to be immunologically distinct; that is, antiserum to each P-form is found to cross-react only very weakly Gth the other (9). The effect of the I’ to S conversion on the subunit interactions has been reported previously (7, S), and is also documented in this report’. That is, t,he S-forms t)end t,o remain as the 50,000 MW monomer under many conditions where the I’-form associates to the 100,000 MW dimer. It was seen that in 0.05 M potassium phosphate, pH 7.0, I’-11 had a molecular weight of about 103,000, while that for S-II in the same solvent \vas about 52,000. The nature of t’he chtbmical change involved in the P to S conversion is the subject of the following paper (25). ACKNOWLEDGMENTS The collaboration of Dr. Frances C. Wornack and Miss Kate Welch is gratefully acknowledged. REFERENCES 1. MCDONALD, M. (1955) Methods Enrymol. 1, 269. 2. MARROW, R. A., AND COLOWICK, ti. P. (1962) Methods Enzymol. 6, 226. 3. TRAYSPR, K. A., AND COLOWICK, S. I?. (1961) Arch. Biochem. Biophys. 94, 177. 4. KAJI, A., TRAYSF,R, K. A., AND COLOWCK, S. P. (1961) Ann. X. I-. Acad. Sci. 94, 798. 5. KENKARE, U. W., SCHULZE, I. T., GAZITH, J., AND COLOWICK, S. P. (1964) A&t. Fifth Intern. Gong. Biochem. IUB. 32, 477. 6. GAZITH, J., SCHULZE, I. T., GOODING, 11. H., WOMACK, F. C., AND COLOWICK, S. P. (1968) Ann. N. Y. Acad. Sci. 161, 307. 7. SCHULZE, I. T., AND COLOWICK, S. P. (1969) J. Biol. Chem. 244, 2306.

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11. 12. 13. 14.

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745. 15. SCHMIDT, J. J., AND COLOWICK, S. P. (1970) Fed. Proc. 29,334, 16. DERECHIN, M., RAMEL, A., LAZARUS, N. R., AND BARNARD, E. A. (1966) Biochemistry 6,

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20. COLOWICK, S. P., WOMACK, F. C., AND NIELSEN, J. (1969) in The Role of Nucleotides for the Function and Conformation of Enzymes (Kalckar, H. M., ed.), p. 15, Alfred Benzon Sympoisum, 1968, Munksgaard, Copenhagen. 21. WARBURG, O., AND CHRISTIAN, W. (1943) Biochem. 2. 310, 384. 22. LAYNE, E. (1957) Methods

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Y. V., AND SCHERAGA, H. A. (1962) Biochemistry 1, 698. 28. CRESTFIELD, A. M., MOORE, S., AND STEIN, W. H. (1963) J. Biol. Chem. 238,622. 29. MOORE, S. (1963) J. Biol. Chem. 238, 235. 30. EDELHOCH, H., KATCHALSKI, E., MAYBURY, R. H., HUGHES, W. L., AND EDSALL, J. T. (1953) J. Amer. Chem. Sot. 76, 5058. 31. MOORE, S., AND STEIN, W. H. (1963) Methods Enzymol. 6, 819. 32. GOODWIN, T. W., AND MORTON, R. A. (1946) Biochem. J. 40, 628. 33. EDELHOCH, H. (1967) Biochemistry 6, 1948. 34. MATSUBARA, H., AND SASAKI, R. M. (1969) Biochem. Biophys. Res. Commun. 36, 176. 35. POTTS, J. T. (1967) Methods Enzymol. 11, 652. 36. BENNET, J. C. (1967) Methods Enzymol. 11,

330. 37. PAULY, H. (1904) 2. fur Physiol. Chemie 42, 508. 38. SMITH, I. (1960) Chromatographic and Electrophoretic Techniques, Vol. 1, p. 296, Interscience Publishers, New York. 3, 297. 39. YPHANTIS, T. (1964) Biochemistry 40. COHN, E. J., AND EDSALL, J. T. (1943) Proteins, Amino Acids, and Peptides, p, 370, Reinhold Publishing Co., New York. 41. CHERVENKA, C. H. (1969) A Manual of Methods for the Ultracentrifuge, Beckman Instruments, Inc., Palo Alto, Calif. 42. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82,70. 43. AMBLER, R. P. (1967) Methods Enzymol. 11, 445. 44. ULLMAN, A., GOLDBERG, M. E., PERRIN, D., AND MONOD, J. (1968) Biochemistry 7, 261. 46. THOMAS, J. O., AND EDELSTEIN, S. J. (1971) Biochemistry

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