Solubilization,purification, and properties of rabbit brain hexokinase

More than 90% of the total hexokinase activity in rabbit brain was found to be associated with the mitochondrial fraction. The participate enzyme was solubilized in a relatively specific way by glucose 6-phosphate and Triton X-100 and purified to apparent homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and affinity chromatography. The solubilized hexokinase activity has been purified 700-fold in 48% yield with a specific activity of 165 units/mg of protein. The molecular weight was found to be approximately 100,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.3 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses with different affinities. Mg · ATP, among the nucleotide substrates, was the most effective as a phosphate donor. The present results indicate considerable similarity between this enzyme and the other mammalian type I hexokinases.     

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme. Effects of flavonoids.

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.     

Pre-steady-state kinetics of beef heart mitochondrial ATPase

The pre-steady-state kinetics of beef heart mitochondrial ATPase (F1) were examined. F1 was found to exhibit hysteretic behavior when hydrolyzing ATP. The hysteretic property was expressed as an activation process which occurred when the enzyme was mixed with its substrate, MgATP. Many catalytic turnovers were required before the activation was complete. The lag in hydrolysis increased hyperbolically as the concentration of enzyme increased. Passage of F1 through Sephadex G25 eliminated the activation process. Several kinetically distinct possibilities for explaining these data, including multiple nucleotide dissociations, enzyme conformational changes, and regulatory site interactions, are discussed. The enzyme was apparently able to recognize nucleotide in a noncatalytic manner, as evidenced by the fact that F1 preincubated with ADP in the absence of substrate achieved partial activation (smaller lag times) before being introduced to substrate. ADP is also a time-dependent inhibitor, exhibiting a slow hysteretic inhibition in addition to immediate competitive inhibition.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

Control of heart mitochondrial oxygen consumption by creatine kinase: The importance of enzyme localization

The route of movement of ADP produced in the mitochondrial creatine kinase reaction was investigated by recording the rate of ADP-dependent oxygen consumption in the presence of phosphoenolpyruvate and pyruvate kinase. This pyruvate kinase system completely abolished activation of respiration by ADP added or by ADP produced in the hexokinase reaction in the medium, but was not able to inhibit the creatine kinase activated respiration when creatine kinase was bound to the inner mitochondrial membrane. These different responses of oxidative phosphorylation were observed at equal $\text{ATP}\text{ADP}$ ratios in the medium. The data obtained evidence direct channeling of ADP from heart mitochondrial creatine kinase to the adenine nucleotide translocase without its prompt release into the medium.     

Chemistry and subunit structure of yeast hexokinase isoenzymes

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.     

Solubilization and characterization of cardiac sarcolemmal taurine-binding proteins

Cardiac sarcolemma preparations of both pig and rat ventricles were found to possess two sets of taurine-binding components. The two proteins from pig heart were solubilized with the detergent Ammonyx-Lo. Characterization of these solubilized proteins revealed that both components are glycoproteins and retain the binding properties observed for the membrane isolate. However, the characterization also revealed several differences between the proteins including their binding specificities, their affinities for taurine, their binding isotherms, and their molecular sizes. Possible functions of these two taurine-binding proteins are discussed.     

Solubilization and condensed packaging of nucleic acids in reversed micelles

RNA and DNA can be solubilized without denaturation in isooctane solutions with the help of reverse micelles formed by di(2-ethyl-hexyl) sodium sulfosuccinate and small amounts of water (down to 0.5%, v:v). With respect to aqueous solutions, RNA (mol. weight 20,000–30,000) in the hydrocarbon micellar solutions shows a decreased absorbance in the 260 nm region, accompanied by an increase of ellipticity. This is attributed to a higher conformational rigidity of the guest biopolymer, and most probably to an increase of base pair stacking. While spectra of low molecular weight samples of DNA (ca. 5000 Daltons) show practically no difference with respect to water, the CD spectrum of the 250,000 Daltons sample is dramatically changed and becomes reminiscent of that of the condensed ψ form. The above spectroscopic effects can be continuously modulated by changing the water content of the micellar system. This offers the possibility of using DNA-containing reverse micelles as models for condensed packaging of DNA in vivo (as in certain phage heads or chromatin).     

Rabbit muscle glycogen-bound phosphoprotein phosphatases: substrate specificities and effects of inhibitor-1

A phosphoprotein phosphatase which has an apparent molecular weight of 240,000 was partially purified (500-fold) from the glycogen-protein complex of rabbit skeletal muscle. The enzyme exhibited broad substrate specificity as it dephosphorylated phosphorylase, phosphohistones, glycogen synthase, phosphorylase kinase, regulatory subunit of cAMP-dependent protein kinase, and phosphatase inhibitor 1. The phosphatase showed high specificity towards dephosphorylation of the beta-subunit of phosphorylase kinase and site 2 of glycogen synthase. With the latter substrate, the presence of phosphate in sites 1a and 1b decreased the apparent Vmax, perhaps by inhibiting the dephosphorylation of site 2. The phosphorylated form of inhibitor 1 did not significantly inhibit this high-molecular-weight phosphatase. However, an inhibitor 1-sensitive phosphatase activity could be derived from this preparation by limited trypsinization. Furthermore, greater than 70% of the phosphatase activity in skeletal muscle extracts and in the glycogen-protein complex was insensitive to inhibitor 1. Limited trypsinization of each fraction obtained from the phosphatase purification increased the total activity (1.5- to 2-fold) and converted the enzyme into a form which was inhibited by inhibitor 1. The results suggest that inhibitor 1-sensitive phosphatase may be a proteolyzed enzyme.     

Solubilization of 20S acetylcholinesterase from chick retina

A 20S form of acetylcholinesterase has been solubilized from young chick retinas by means of a buffered salt-detergent solution containing EDTA. The release of this fast-sedimenting form of the enzyme is selectively blocked by the presence of even small amounts of Ca⁺⁺ in the homogenization medium. The collagen-tailed nature of this molecular species of acetylcholinesterase has been ascertained by collagenase digestion. This finding suggests that the avian central nervous system contains asymmetric, collagen-tailed quaternary structural forms of acetylcholinesterase as is the case in skeletal muscle and cholinergic ganglia.     

Action of 5-thio-D-glucose and its 1-phosphate with hexokinase and phosphoglucomutase.

Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a K_m of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a K_i of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a K_m of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a K_i of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions.     

Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin

Limited treatment of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of the Mr 98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates of Mr 60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as where T1 and T2 indicate tryptic cleavage sites; cleavage at only T1 or T2 gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping using Staphylococcus aureus V8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (approximately 20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on the Km for Glc or the Ki for Glc-6-P, though a slight decrease in Km for ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.     

Reye's syndrome: plasma-induced alterations in mitochondrial structure and function

Preincubation of rat liver mitochondria with plasma from Reye's syndrome (RS) patients induces a transient stimulation of the State 4 respiratory rate of the oxidation of NAD-linked substrates which is followed by inhibition. A loss of nearly 90% of the intramitochondrial NAD⁺ and NADP⁺ is also seen. The respiratory rate cannot be stimulated upon subsequent addition of ADP, but can be fully restored upon the addition of either NAD⁺ or succinate (plus rotenone). The degree of effectiveness depends on the incubation time and the ratio of RS-plasma/mitochondrial protein. The RS-plasma effects can be eliminated by an inhibitor of mitochondrial Ca²⁺ transport (ruthenium red) or by a Ca²⁺ chelator (ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid). Control plasma at a concentration of 2 mg dry wt per milligram of mitochondrial protein, or 30 μm Ca²⁺ gives no effect, but can reproduce the RS-plasma effects completely when a minute amount of allantoin (10^?11 mol/mg mitochondrial protein) is also present. We conclude that allantoin and Ca²⁺ can increase the permeability of mitochondrial membrane, and may be the key components responsible for the mitochondrial injuries produced by RS-plasma.     

Studies on the binding of nucleotides by rat brain hexokinase

Various nucleoside di- and triphosphates have been compared with respect to their ability to protect rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activity against inactivation by chymotrypsin, glutaraldehyde, heat, and 5,5′-dithiobis(2-nitrobenzoic) acid. ATP could be distinguished from other nucleoside triphosphates in these comparisons, which may be related to the specificity with which ATP is utilized as a substrate. All nucleoside derivatives examined provided substantial protection against two or more of the above inactivating agents, indicating relatively nonspecific binding of nucleotides by brain hexokinase, consistent with a similar lack of specificity in the inhibition of this enzyme by nucleoside derivatives. The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tetraiodofluorescein (TIF) was enhanced by binding to brain hexokinase. TNS binding was not affected by the presence of various relevant metabolites (Glc, glucose 6-phosphate, ATP), nor did TNS inhibit the enzyme. In contrast, substantial (approximately 70%) decreases in the fluorescence of bound TIF resulted from the addition of various nucleoside derivatives, and TIF served as a competitive inhibitor of brain hexokinase. These observations are consistent with the view that TIF binds to a nucleotide binding site of the enzyme. The inability of nucleotides to totally displace TIF was taken to indicate the existence of an additional TIF binding site (or sites) discrete from the catalytic site, and probably identical to the site(s) at which TNS binds with no effect on catalytic activity. The effects of saturating levels of ATP and ADP were not additive indicating that both compounds were displacing TIF from the same site i.e., a common nucleotide binding site. Glc, mannose, and 2-deoxyglucose greatly enhanced the ability of nucleotides to displace TIF, while fructose, galactose, and N-acetylglucosamine did not, indicating the existence of interactions between hexose and nucleotide binding sites; the hexoses themselves were not effective at displacing TIF. The enhanced binding of nucleotides in the presence of the first three hexoses but not the latter three can be directly correlated with the relative ability of these hexoses to induce specific conformational changes in the enzyme. The hexoses themselves were not effective at displacing TIF. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate could also displace TIF, and as with the nucleotides, a maximum of approximately 70% decrease in fluorescence was observed and the effectiveness of glucose 6-phosphate was enhanced in the presence of Glc. Other hexose 6-phosphates tested were not effective at displacing TIF. The specificity with which hexose 6-phosphates displaced TIF could be correlated with their ability to induce specific conformational change in the enzyme. The results are discussed as they relate to the kinetic mechanism and allosteric regulation by nucleotides that have been proposed for this enzyme.     

Solubilization and partial purification of the high-affinity gonadoliberin receptor from the bovine pituitary gland

The high-affinity gonadoliberin (GnRH) receptor contained in a membrane preparation from frozen bovine anterior pituitary glands has been solubilized in Triton X-100 and the binding properties of the solubilized product have been examined. The radioreceptor-binding assay, using the GnRH agonist [D-Ser(t-Bu)⁶] des-Gly¹⁰GnRH N-ethylamide (GnRH-A) as radioligand, demonstrated that the kinetics of association and dissociation, the binding constants, as well as the specificity of receptor were not altered in the solubilized receptor preparations. Affinity chromatography on a concanavalin A-Sepharose column, with elution of adsorbed material using a solution of α-methyl-d-mannoside, allowed a 33-fold purification of the receptor. The K_a of the receptor thus purified was of the same order as that of the starting material, although slightly higher values were found. Only about one-half of the total receptor activity applied to the column was retained in spite of several recyclings. The other half was found in the nonadsorbed fraction. It is postulated that the detergent-solubilized fraction contains two forms of the GnRH receptor. The nonadsorbed fraction probably contains a partially or totally deglycosylated form. It is possible that the detergent-solubilization process somewhat alters the physicochemical properties of a part of the GnRH receptor molecules. Electrophoretic analysis of the purified receptor preparations, with a subsequent GnRH-binding assay, suggests that the apparent molecular mass of the high-affinity GnRH receptor, or of its monomeric form, is approximately 60,000 Da.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver

The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.