Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation.

Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (K(i)=30 microM) and mannoheptulose (K(i) congruent with 300 microM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The K(i) for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases.     

Differential molecular sieving permits visualization of hexokinase isoenzymes following electrophoretic separation in high resolution acrylamide gels

1. Differential molecular sieving is the concept applied to bring together isoenzymes of ATP:D-hexose-6-phosphotransferase (hexokinases) with glucose 6-phosphate dehydrogenase in acrylamide gels by utilization of their dissimilar electrophoretic mobilities. 2. The hexokinase isoenzymes migrate and separate in gels with pore sizes selected to entrap glucose 6-phosphate dehydrogenase in their interstices. The locations of the bands of specific activity are visualized by fluorescence of NADPH under long wave, ultraviolet radiation. 3. A new discontinuous electrochemical system has been devised to deliver protective thiol groups into the gel. Cysteine (trailing ion) and SO4(2-) (leading ion) form a sharp moving boundary. 4. The high resolution of the system has permitted visualization of a rapidly migrating, high Km hexokinase in murine spleen, fat, kidney and lymph nodes. Hexokinase Types I and II, were observed in all tissues tested, but Type IV was seen only in the liver. 5. The importance of glucose concentration effects on hexokinase activity is emphasized by inactivation of slowly migrating low Km hexokinase Types I and II following exposure to 200 mM glucose during preparation of extracts.     

Purification and properties of suppressor seryl-tRNA: ATP phosphotransferase from bovine liver

Seryl-tRNASerCmCA: ATP phosphotransferase was purified 1200-fold from bovine liver by ultracentrifugation at 150 000 X g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, chromatography on hydroxyapatite, gel filtration on Sephacryl S-300 and affinity chromatography on Blue Sepharose. Molecular mass was estimated as 135-145 kDa. The Km values for ATP and ser-tRNASerCmCA were 2 mM and 21 nM, respectively. This enzyme did not react with ser-tRNASerIGA, tyr-tRNA or thr-tRNA.     

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.     

Allosteric inhibition of brain hexokinase by glucose 6-phosphate in the reverse reaction

A study of the reverse reaction of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been performed using a photometric method based on a mutarotase-glucose oxidase-peroxidase-chromogen system to trap and visualize glucose, plus a glycerol kinase-glycerol system to trap ATP. Glucose 6-phosphate or 2-deoxyglucose 6-phosphate were used as phosphoryl donors at different concentrations of ADP. Variation of glucose 6-phosphate concentrations resulted in a biphasic curve from which apparent Km and Ki values of ca. 0.2 mM were calculated. In contrast, variation of 2-deoxyglucose 6-phosphate concentrations resulted in Michaelian kinetics with an apparent Km of 2 mM. The Km value for MgADP was 16 mM irrespective of the nature and concentration of the hexose 6-phosphate substrate. These results are fully consistent with an allosteric site for glucose 6-phosphate as an explanation for the inhibition of animal hexokinases by glucose 6-P and further indicate that the maximal rate is the parameter affected. From these observations and previous knowledge, the possible occurrence in animal hexokinases of a regulatory site for ATP to account for the competition between glucose 6-phosphate and ATP in the forward reaction is postulated.     

Hexokinases of tobacco leaves: Subcellular localization and characterization

Subeellular localization of the enzymes which phosphorylate hexoses was studied in photosynthesizing tobacco leaves by means of differential centrifugation and centrifugation in sucrose gradient. More than 80 % of the total hexokinase activity of leaf tissues were found to be associated with the particulate fraction of mitochondria; however, the ratio of the particulate hexokinase fraction to the soluble fraction was influenced by the extraction medium applied. The particulate hexokinases showed a high affinity to glucose (Km = 26.8 μM) and a relatively low affinity to fructose (Km = 17.6 mM). They had a broad pH optimum, because 81 % of the phosphorylating activity obtained for glucose and 75 % of the activity obtained for fructose occurred in pH range from 7.9 to 9.1 (Tris-HCl buffer). The hexokinases were Mg2+ dependent with the highest activity occurring at equimolar Mg2+ and ATP concentrations. Their activity was enhanced by KC1, NaCl, and (NH4)2SO4 at 30 to 120 mM concentrations.     

All hexokinase isoenzymes coexist in rat hepatocytes.

The cellular distribution of hexokinase isoenzymes, N-acetylglucosamine Kinase and pyruvate kinases in rat liver was studied. Hepatocytes and non-parenchymal cells with high viability and almost no cross-contamination were obtained by perfusion in situ of the liver with collagenase, with the use of an enriched cell-culture medium in all steps of cell isolation. Separation of hexokinase isoenzymes was done by DEAE-cellulose chromatography, and enzyme activities were measured by a specific radioassay. Cytosol from isolated hepatocytes contained high-affinity hexokinases A, B and C, in addition to hexokinase D. The last-mentioned represented about 95% of total glucose-phosphorylating activity. Only hexokinase A was found associated t the particulate fraction. Isolated non-parenchymal cells contained only hexokinases A, B and C. N-Acetylglucosamine kinase was measured with a specific radioassay and was found as a single enzyme form in both hepatocytes and non-parenchymal cells, with higher activities in the former. Pyruvate kinase isoenzyme L was present only in the hepatocytes and isoenzyme K only in the non-parenchymal liver cells, confirming that they are good cellular markers.     

Hexokinase isoenzymes of RIN-m5F insulinoma cells. Expression of glucokinase gene in insulin-producing cells.

We have analysed the pattern of expression of the hexokinase isoenzyme group in RIN-m5F insulinoma cells. Three hexokinase forms were resolved by DEAE-cellulose chromatography. The most abundant isoenzyme co-eluted with hexokinase type II from rat adipose tissue and displayed a Km for glucose of 0.15 mM, similar to the adipose-tissue enzyme. Hexokinase type II was in large part associated with a particulate subcellular fraction in RIN-m5F cells. The two other hexokinases separated by ion-exchange chromatography were an enzyme similar to hexokinase type I from brain and glucokinase (or hexokinase type IV). The latter isoenzyme was identified as the liver-type glucokinase by the following properties: co-elution with hepatic glucokinase from DEAE-cellulose and DEAE-Sephadex; sigmoid saturation kinetics with glucose with half-maximal velocity at 5.6 mM and Hill coefficient (h) of 1.54; suppression of enzyme activity by antibodies raised against rat liver glucokinase; apparent Mr of 56,500 and pI of 5.6, as shown by immunoblotting after one- and two-dimensional gel electrophoresis; peptide map identical with that of hepatic glucokinase after proteolysis with chymotrypsin and papain. These data indicate that the gene coding for hepatic glucokinase is expressed in RIN-m5F cells, a finding consistent with indirect evidence for the presence of glucokinase in the beta-cell of the islet of Langerhans. On the other hand, the overall pattern of hexokinases is distinctly different in RIN-m5F cells and islets of Langerhans, since hexokinase type II appears to be lacking in islets. Alteration in hexokinase expression after tumoral transformation has been reported in other systems.     

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.     

Inhibition and inactivation of glucose-phosphorylating enzymes from Saccharomyces cerevisiae by D-xylose

Three glucose-phosphorylating enzymes were separated from cell-free extracts of Saccharomyces cerevisiae by hydroxylapatite chromatography. Variations in the amounts of these enzymes in cells growing on glucose and on ethanol showed that hexokinase PI was a constitutive enzyme, whereas synthesis of hexokinase PII and glucokinase were regulated by the carbon source used. Glucokinase proved to be a glucomannokinase with Km values of 0.04 mM for both glucose and mannose. D-Xylose produced an irreversible inactivation of the three glucose-phosphorylating enzymes depending on the presence or absence of ATP. Hexokinase PI inactivation required ATP, while hexokinase PII was inactivated by D-xylose without ATP in the reaction mixture. Glucokinase was protected by ATP from this inactivation. D-Xylose acted as a competitive inhibitor of hexokinase PI and glucokinase and as a non-competitive inhibitor of hexokinase PII.     

Characterization and compartmentation,in green leaves,of hexokinases with different specificities for glucose,fructose, and mannose and for nucleoside triphosphates

When green leaves of spinach (Spinacia oleracea L.) were surveyed for the presence of hexokinases which utilize glucose, fructose and-or mannose as a substrate, four kinases could be distinguished by their order of elution during chromatography on diethylaminoethyl (DEAE)-cellulose: (i) a hexokinase I with a specificity for fructose, glucose, and mannose, (ii) a fructokinase I with a specificity for fructose, (iii) a hexokinase II with a specificity for glucose, fructose and mannose, and (iv) a fructokinase II with a specificity for fructose. Hexokinases I and II had high apparent K_m values for fructose (8 and 15 mM, respectively) and medium or low apparent K_m values for glucose (150 and 18 μM, respectively) and mannose (18 and 15 μM, respectively). Maximal velocities were highest with fructose, medium with glucose and lowest with mannose. That hexokinases I and II used several sugars as substrate was concluded (i) from their identical elution profiles during enzyme separation and (ii) because their activities with two or three sugars at a time was always lower than the sum of activities with one substrate, indicating competition of the sugars for the reaction with the enzymes. Fructokinases I and II were very specific for fructose (85 and 140 μM, respectively) and had only little, if any, activity with glucose or mannose. All kinases showed varying degrees of activity with nucleoside triphosphates other than ATP. In the presence of all three sugars, hexokinases I and II were considerably more active with ATP than with uridine-, cytidine-, and guanosine 5'-triphosphate (UTP, CTP, GTP) except that, in the presence of glucose, hexokinase I was almost as active with UTP as with ATP. In the presence of fructose, fructokinase I exhibited highest activity with GTP and a gradually decreasing level of activity with CTP, UTP, and ATP. The activities in the presence of the other two sugars were highest with ATP. Fructokinase II was most active with ATP and fructose and progressively less active with GTP, UTP, and CTP. Cell fractionation by isopycnic density-gradient centrifugation or differential centrifugation indicated that fructokinase II was associated with chloroplasts, hexokinase II with mitochondria, and the other two kinases with the non-particulate cell fraction. In green leaves of pea (Pisum sativum L.), only a hexokinase (II) and fructokinase (II) were present. Corn (Zea mays L.) leaves exhibited only very low hexokinase activity. Dedicated to Prof. Dr. Hans Mohr on the occasion of his 60th birthday     

The Preparation of Yeast Hexokinases

Improved methods are described for the preparation of hexokinase from baker's yeast. The isolation procedure is designed to avoid proteolysis, by using mechanical disintegration of the yeast cells, by organophosphate inhibition of the serine-dependent proteases, and by removal of all other proteases by gel filtration.

Three isoenzymes, A, B and C, can be obtained thus. For hexokinase A, the ratio of activity in phosphorylating fructose as compared to glucose is about twice that of B or C. Hexokinase C is very similar in properties to B, but is separable by ion-exchange chromatography and appears to be a conformational isoenzyme of B. In the final purified state, the specific activity on glucose (27 mM, pH 8.3, 25.0) is 275 international units per mg for A, 900 for B and 750 for C, these values being higher than those for previously reported forms.     

Purification and Properties of Purified Hexokinase from the African Trypanosomes and Trypanosoma equiperdum

SYNOPSIS. Hexokinase was in both soluble and particulate fractions of extracts of Trypanosoma brucei, T. gambiense, T. rhodesiense and T. equiperdum. These enzymes have been purified some 350-fold from crude extracts by sonication, (NH₄)₂SO₄ fractionation, and DEAE Sephadex column chromatography. Purified hexokinases had: A) temperature optimum 45-50 C; B) pH optimum 6.5-7.0; C) Mg⁺⁺ and ATP requirements; D) inhibition by ADP, p -hydroxymercuribenzoate, glucose-6-phosphate, and competitive inhibition by mannose, glucosamine, N -acetyl-D-glucosamine and xylose; E) ability to phosphorylate glucose, fructose, mannose, 2-deoxy-D-glucose and glucosamine; F) a glucose requirement for stabilization. T. equiperdum hexokinase was inhibited 33% by ADP as compared to 17-20% with brucei group hexokinase, and showed 22% activity when ITP was substituted for ATP in contrast to 35-40% with brucei group hexokinase.     

Hexokinase of Angiostrongylus cantonensis: presence of a glucokinase.

1. Angiostrongylus cantonensis contains a glucokinase which was isolated by DEAE-cellulose column chromatography. 2. This enzyme has a much higher affinity toward glucose (apparent Km, 0.2 mM) than fructose (apparent Km, 85 mM). Glucose-6-phosphate (10 mM) does not inhibit glucose phosphorylation. 3. Molecular weight obtained by a molecular sieve chromatography (60,000) is also close to the value of mammalian glucokinase. 4. While Vmax value for mannose is one-third smaller than that for glucose, Km for mannose is rather lower than that for glucose. 5. In addition to the cytosol enzyme, a particle bound hexokinase is found in the worm.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Purification and properties of beta-galactosidase from chicken seminal plasma

1. beta-Galactosidase (EC 3.2.1.23) from chicken seminal plasma was purified approx. 111-fold to homogeneity. 2. pH optimum of the enzyme ranged from 3.6 to 4.0 and its Km was 0.65 mM with p-nitrophenyl-beta-D-galactoside as substrate. 3. The enzyme was unstable at its optimal activity pH and was activated by Cl- ions. 4. The enzyme had pI value of 4.0. 5. The active enzyme had Mr approx. 100,000 by Sephacryl S-300 chromatography. SDS electrophoresis in the presence of beta-mercaptoethanol showed four bands corresponding to Mr of approx. 90,000, 75,000, 65,000 and 13,000.     

Hexokinase activity alters sugar-nucleotide formation in maize root homogenates

Two pools of hexokinase activities differing in sensitivity to ADP inhibition were characterised in maize roots. In order to evaluate how glucose utilisation could be affected by these hexokinases, glucose-6-P and NDP-5'-sugar levels were measured after a D-[U-14C]glucose pulse in root extracts in the presence of 0 or 1 mM ADP. Analysis of radio-labelled activated sugars by paper chromatography revealed that: (1) without ADP, nearly 20% of the 14C appeared in NDP-5'-sugars; (2) 0.1 mM ADP inhibited 14C-NDP-5'-sugar formation by 85%; and (3) with 1 mM ADP, 14C-NDP-5'-sugars were undetectable, but substantial (14%) 14C accumulated as glucose-6-P. Mannoheptulose, a hexokinase inhibitor, blocked the NDP-5'-sugar formation, but did not modify the amount of 14C-glucose-6-P in root extracts either with or without ADP. The analysis of the hexokinase activities with 0.8 mM glucose in maize root extracts showed that: (1) mitochondrial hexokinase activity was totally inhibited by 30 mM mannoheptulose; and (2) the cytosolic hexokinase was inhibited by only 30%. These data suggest that NDP-5'-sugar synthesis is sensitive to ADP fluctuations and that mannoheptulose affects preferentially the mitochondrial-bound hexokinase, but the cytosolic form is less sensitive. We propose that the mitochondrial hexokinase is the main energy charge sensor in this pathway in maize.     

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.     

Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo

A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.     

Cloning and biochemical characterization of a novel mouse ADP-dependent glucokinase

Glycolysis, the catabolism of glucose to pyruvate, is an iconic central metabolic pathway and often used as a paradigm for explaining the general principles of the regulation/control of cellular metabolism. The ubiquitous mammalian ATP-dependent hexokinases I-III and hexokinase IV, also termed glucokinase, initiate the process by phosphorylating glucose to glucose-6-phosphate. Despite glycolysis having been studied extensively for over 70 years and the last new mammalian ATP-dependent hexokinase isotype having been described in the 1960s, we report here the biochemical characterization of a recombinant ADP-dependent glucokinase cloned from a full-length Mus musculus cDNA, identified by sequence analysis. The recombinant enzyme is quite specific for glucose, is monomeric, has an apparent Km for glucose and ADP of 96 and 280 microM, respectively, and is inhibited by both high concentrations of glucose and AMP. The metabolic role of this enzyme in cells would be dependent on the relative level of its activity to those of the ATP-dependent hexokinases. The greatest advantage of an ADP-GK would clearly be during ischemia/hypoxia, clinically relevant conditions in multiple major disease states, by decreasing the priming cost for the phosphorylation of glucose, saving ATP.