Sidney P. Colowick

Isolation of skeletal muscle cell membrane and some of its properties.

A procedure is described for the isolation of empty tubular cell membrane-like structures from rat skeletal muscle. The procedure resulted in a purification of about 140-fold in terms of protein content per tube. The yield was about 20% in terms of total number of tubes recovered. The purest preparations contained about 65% protein, 15% lipid, and 3% carbohydrate on a dry-weight basis. Most of the lipid was phospholipid which became ether-extractable only on acidification. Most of the protein had the composition of collagen. That the membrane structure itself contained a collagen-like protein is suggested by the fact that the membranes were dissolved by collagenase or dilute acetic acid and showed a critical shrinkage temperature at 57–59°. A preliminary survey of enzyme activities found in the membrane fraction is reported. No evidence was obtained for binding of insulin or d -galactose by the purified membrane fraction.

MULTIPLE FORMS AND SUBUNITS OF YEAST HEXOKINASE

In a previous symposium of this Academy on multiple molecular forms of enzymes, a paper from this laboratory was presented (Kaji et al., 1961), in which multiple forms of yeast hexokinase were described. Since that time, it has been found that most, if not all, of these forms were derived by the action of a protease acting during the course of the preparation. In the present paper, the nature of this modification process is described, and the chemical, physical, and catalytic properties of unmodified and modified enzyme are summarized. Evidence is presented that, when proteolytic modification is avoided, two distinct forms of yeast hexokinase can be demonstrated, with different catalytic and chemical properties. The method for preparing these unmodified forms has been described elsewhere (Schulze et al., 1967). Similar results have been obthined independently in Barnards laboratory (Lazarus et al., 1966 and Derechin et al., 1966).

Chemistry and subunit structure of yeast hexokinase isoenzymes.

Abstract 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.

Purification and serological comparison of the yeast hexokinases P-I and P-II.

Abstract An improved procedure for purification of the hexokinases P-I and P-II from bakers yeast is described. Yields, reproducibility, and purity are improved over those found by the methods used previously in this laboratory. The growth of large crystals of form P-I is described. Antisera prepared against the two purified hexokinases show only slight cross reaction by microcomplement fixation. The anti-sera have been used to demonstrate the presence of both P-I and P-II in crude extracts of various yeasts, including two haploid strains, and their absence in a yeast which contains glucokinase but no hexokinases.

Identification of a peptide sequence involved in association of subunits of yeast hexokinases

Abstract The chemistry of the proteolytic conversion of the native yeast hexokinases P-I and P-II to the respective modified forms S-I and S-II was studied in detail. The conversion of P-I to S-I was found to involve the removal of one six and one five residue peptide from P-I; these peptides were isolated and sequenced, and a comparison with the partial sequence of native P-I demonstrated that they were cleaved from the amino terminal end. Since results indicated that exactly the same peptides were cleaved from P-II during conversion to S-II, it is concluded that the first 11 amino acids in P-I and P-II have the same sequence. That sequence is: val · his · leu · gly · pro · lys · lys · pro · gln · ala · arg The basicity of these peptides was reflected in the decrease in isolectric point observed when a P-form is converted to an S-form. The peptides are clearly involved in the association of the subunits of yeast hexokinase, since their removal greatly weakens the tendency of the subunits to dimerize.

Catalytic activity with associated and dissociated forms of the yeast hexokinases

Abstract In agreement with previous glucose binding data, kinetic studies of the yeast hexokinases at high protein concentration show that the dimeric forms P-I and P-II require much higher glucose concentrations for half-maximal rates than do the monomeric forms S-I and S-II, but each P form reaches the same V value as the corresponding S form. Raising the temperature from 5 °C to 24 °C causes an apparent dissociation of the P forms, most pronounced in the case of P-I, as evidenced by a lowering of glucose K M values toward those of the S forms. Similarly, kinetic measurements at low protein concentrations, where the P forms are presumably fully dissociated, give identical glucose K M values for the P and S forms.

Aminoethyl phosphoric ester in the small intestine of rabbits and pigs.

Summary A fractionation of the acid-soluble organic phosphoric esters of the small intestine as the uranium salts has been described. Aminoethyl phosphoric ester and adenylic acid have been isolated in crystalline form, each comprising about 1/4 of the acid-soluble organic P of the rabbit small intestine. The occurrence of the former ester in pig intestine has also been demonstrated.

[38] Growth factor stimulation of sugar uptake

Publisher Summary In an investigation of factors that regulate metabolic events, it is important to examine the relationship of membrane transport processes and intracellular substrates at rates that are consistent with metabolic activity. Glucose transport in most nonepithelial cells occurs via the process of facilitated diffusion that involves the establishment of equilibrium across the plasma membrane. This mechanism can be subcategorized into two systems: nonregulated and regulated transport. Nonregulated transport is a characteristic of red blood cells, hepatocytes, and brain cells. Regulated transport, which gives the system the ability to adjust to cellular requirements, is observed in cells such as fibroblasts, adipocytes, and muscle fibers. This chapter describes the procedure for determining the rate of glucose uptake into monolayer mouse Swiss 3T3 fibroblast cells in culture. The chapter describes a specific application of the method in which the stimulation of glucose uptake occurs by a transforming growth factor α (TGFα) epidermal growth factor (EGF) and transforming growth factor β (TGFβ).

Purine nucleoside diphosphate regulation of yeast hexokinases

Abstract The low activity state of hexokinase P-II, originally produced by Kosow and Rose by lowering the pH from 8 to 7 in certain sulfonated buffers, is not observed in Tris or imidazole buffers at pH 7 unless low concentrations of ADP or GDP are added. At pH values below 7 in imidazole buffer, partial inhibition occurs by protonation alone, and ADP or GDP causes further inhibition. As in the Kosow-Rose experiments, the enzyme in the low activity state can be activated either by excess ATP or by low concentrations of citrate, 3-phosphoglycerate and other metabolites. The inhibition by nucleoside diphosphates is greater at high glucose concentration. Hexokinase P-I is much less susceptible to regulation by nucleoside diphosphates or citrate, suggesting different physiological roles for the two isoenzymes.

The increase in hexokinase activity in heredityry avian muscular dystrophy

Hexokinase activity was found to be increased in both the more severely affected red (thigh) muscle of dystrophic chickens. The increase in activity was largely associated with the particulate fraction. These findings may indicate early events in the pathogenesis of avian muscular dystrophy.