Michael A. Rosemeyer

The molecular weight and dissociation properties of yeast hexokinase

Hexokinase (ATP: D-hexose 6_phosphotransferase, EC 2.7.1.1) is the first enzyme of the glycolytic pathway and as such is of critical importance to the overall energy-producing metabolism of the cell. Its key position in glycolysis also suggests that it may be subject to metabolic control by allosteric effecters. Before such phenomena can be assessed it is necessary to determine the number of subunits of the enzyme and to investigate their interactions. The present research attempts to establish the molecular weight of the enzyme and to characterise the various dissociation equilibria in which it is involved. It also shows that, owing to the dissociation phenomena, discrete molecular species were seldom observed. Hexokinase was purified from yeast by Berger, Slein, Colowick and Cori [l] in 1946, and in the same year by Kunitz and McDonald [2]. The latter authors were the first to estimate the molecular weight as 96,600 in acetate at pH 5.5. Subsequent preparations have provided evidence that multiple forms of the enzyme occur [3]. Some of these forms appear to be produced by proteolytic modification of the native hexokinases, and differ from them in molecular and electrophoretic properties. Recently Lazarus, Derechin and Barnard [4] have reported molecular weights of 102,000 and 104,000 in acetate buffer, pH 5.5 10.1. Hexokinase was isolated from baker’s yeast (Easterby and Rosemeyer, unpublished work). Each of the proteins obtained was homogeneous on starch gel electrophoresis and ultracentrifugation. During the purification, addition of phenylmethyl sulphonylfluoride (PMSF) to protect against proteolysis [ 51 resulted in two forms of the enzyme, designated hexo-

Evidence for the monomeric nature of thymosins

According to gel‐filtration experiments, α‐ and β‐thymosins appear to form oligomers, which are 4‐5‐fold larger than the corresponding polypeptides. However, on analysis by sedimentation equilibrium ultracentrifugation, prothymosin α and thymosin β4 showed relative molecular masses of 12 800 and 4600, which are close to the values calculated from their amino acid sequences, confirming their existence in solution as discrete monomeric entities.

On the molecular size of thymosins

The immunoregulatory polypeptide prothymosin α and its biologically active N‐terminal fragment thymosin α1m, with relative molecular masses of 12 500 and 3108 respectively, were found to behave as oligomers (trimers to hexamers) in gel‐filtration measurements. This phenomenon of an apparent association of polypeptides has been reported for other thymosins — parathymosin α, thymosin β4 and thymosin β10. In contrast, sedimentation equilibrium ultracentrifugation shows that thymosin α1 is a monomer with a relative molecular mass of 3000±200. Measurement of the diffusion coefficient as 221 μm2/s suggests that the molecule is approximately spherical. The implications for the molecular species of prothymosin α, parathymosin α, and β‐thymosins are discussed.

The molecular weight and subunit structure of glucose-6-phosphate dehydrogenase from human erythrocytes

Glucose&phosphate dehydrogenase (G6PD; EC 1 .l .1.49) catalyses the initial reaction of the pentose phosphate pathway, and thus its function is necessary in maintaining the level of the reduced coenzyme NADPH in various tissues. Human G6PD exhibits polymorphism, the structural gene being on the X-chromosome [ 11. The variant forms differ from the normal enzyme in electrophoretic mobility or in catalytic activity [2,3]. While deficiencies in function do not appear to affect other tissues, most variants with low activity are associated with haemolytic anaemias, either chronic or induced by administration of drugs or ingestion of toxic substances. In the red blood cell, NADPH is used to reduce glutathione [4], which is required to preserve sulphydryl groups and so keep the cell intact. On the basis of kinetic measurements, it has been suggested that catalysis by G6PD in the red cell and in other tissues may be subject to metabolic control [S-l 01. In studying this possibility, or the extent of such control, it is necessary to know the molecular size and number of subunits of the enzyme. Previous molecular weight measurements of the normal erythrocyte enzyme, termed Gd(+) B [2], varied between 105,000 [ 1 l] and 240,000 [ 121. Yoshida concluded from the molecular weight in 4 M guanidium chloride that the enzyme contains 6 subunits [ 123, and from the number of tryptic peptides that the subunits were identical [ 131. In the present investigation of the enzyme, the sedimentation coefficient was found to vary with the solvent conditions. The data also indicate that more than one dissociation equilibrium was occurring. It is necessary to separate these equilibria to establish the molecular size and subunit content of the protein.

Sickle cell disease: the proportion of liganded haemoglobin needed to prevent crises.

Summary. In an attempt to predict the likelihood of successfully treating sickle cell disease by increasing haemoglobin S (Hb S) oxygen affinity, two liganded derivatives of Hb S have been studied in an in vitro system that measures deoxy‐Hb S polymerization. The participation of these liganded forms in the polymers has been quantitated in terms of an exclusion factor that relates their behaviour to that of deoxy‐Hb S.

Isolation and glucose-6-phosphate-mediated dimerization of hexokinase from human heart.

Soluble hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from human heart. 1 kg of tissue provided 25 mg hexokinase with a specific activity of 58 units/mg, representing a 1700-fold purification and 47% yield. The purification involved six steps, including affinity chromatography with glucosamine attached to Sepharose. The material was homogeneous according to electrophoresis, gel-filtration and sedimentation in the ultracentrifuge, but gave two main components on electrophoresis in denaturing conditions. From determination of the sedimentation and diffusion coefficients, the relative molecular mass was calculated to be 105 000. The enzyme is monomeric, but glucose 6-phosphate promotes an association to dimers. This effect is reversible and is independent of the concentrations of glucose or inorganic phosphate. The results support the postulate that soluble and mitochondrion-bound hexokinases are identical.

Purification and physical properties of hexokinase from human erythrocytes.

A bulk purification is described for hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from human erythrocytes. Following a 110,000-fold purification from 40 litres of blood, 5 mg protein with a specific activity of 22 units/mg were obtained. On application of various separation techniques, the enzyme activity co-migrated with the main protein component. The physical properties, such as the relative molecular mass of 108,000 and sedimentation coefficient of 5.5 S, are similar to those of the enzyme from human heart. In particular, there is a correspondence in the conformational response to glucose 6-phosphate as shown by an association of the enzyme promoted by this metabolite.

Microtubule formation and the initial association of tubulin dimers

Microtubule protein could be prepared in high yield, and could form copious microtubules, in solutions containing glutamate but not in solutions containing only phosphate ions. Correspondingly, tubulin after isolation showed an association equilibrium in the presence of glutamate (or other zwitterions), but not in phosphate buffers. The correlation suggests that this association to tetramers is probably the initial step in the mechanism of microtubule formation.

Increasing haemoglobin oxygen affinity to prevent sickling: abnormal haemoglobin variants as models

Summary. TWO abnormal high oxygen affinity haemoglobins have been studied for their ability to inhibit the polymerization of deoxy‐Hb S. They were used as models to predict the effect of chemically modifying haemoglobin to increase oxygen affinity since the oxy(R)‐conformation is the most potent inhibitor known of cell sickling. The participation of these variants in deoxy‐Hb S polymers has been described quantitatively in terms of an exclusion coefficient, f, that relates their behaviour to that of deoxy‐Hb S, and qualitatively in terms of an exponent of hybridization.

The correlation between the apparent molecular weight and the enzymic activity of lactose synthetase

This catalysis is referred to as lactose synthetase activity, or AB activity. Protein B is a-lactalbumin [3] present in large amounts in milk [4] . Methods for isolating the galactosyl transferase rely on retention of the enzyme, in the presence of N-acetylglucosamine [5] or glucose [6] , by a-lactalbumin which is covalently-bound to an inert matrix. The procedure is indicative of an association between the two proteins. In the initial stages of purification of the galactosyl transferase, it is separated from excess cy-lactalbumin by gel-filtration. Repeated gel-filtration gave various elution volumes for the enzyme, presumably depending on the amount of o-lactalbumin still complexed with the transferase [7]. It should be possible to assess the relative amounts of ar-lactalbumin present in the eluate by measuring the ability to catalyse each of the two reactions. Work in this laboratory [8] has shown that a com-