In vitro synthesis of rat brain hexokinase

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)+ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipitation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical 'soluble' enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.     

In vitro synthesis of rat brain hexokinase

Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)⁺ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipatation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical ‘soluble’ enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that (1) it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and (2) limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.     

Difference in hydrophobicity between mitochondria-bindable and non-bindable forms of hexokinase purified from rat brain

Hexokinase able to bind to mitochondria was purified to homogeneity from rat brain by two successive DEAE-cellulose chromatographic steps. The enzyme lost only the binding ability with almost undetectable change in molecular weight on mild chymotrypsin digestion. The bindable hexokinase was adsorbed to a Phenyl-Sepharose column and eluted with a Lubrol PX gradient, whereas non-bindable hexokinase and yeast hexokinase were not adsorbed under the similar conditions. These results suggest that mitochondria-bindable hexokinase has a hydrophobic region on its surface, which is responsible for the specific interaction with mitochondria.     

Biophysical investigations on the active site of brain hexokinase

Replacement of Mg (II), the natural activator of brain hexokinase (EC 2.7.1.1) by paramagnetic Mn (II) without affecting the physiological properties of the enzyme, has rendered brain hexokinase accessible to investigations by magnetic resonance methods. Based on such studies, a site on the enzyme, where Mn (II) binds directly with high affinity has been identified and characterized in detail. Use ofβ,γ-bidentate Cr (III) ATP as an exchange-inert analogue for Mn (II) ATP has shown that Mn (II) binding directly to the enzyme has no catalytic role but another Mn (II) ion binding simultaneously and independently to the enzyme through the nucleotide bridge participates in enzyme function. However, using this direct binding Mn (II) ion and a covalently bound spin label as paramagnetic probes a beginning has been made in mapping the ligand binding sites of the enzyme. Ultra-violet difference spectroscopy has revealed the presence of at least two glucose 6-phosphate locations on the enzyme one of which presumably is the high affinity regulatory site modulated by substrate glucose. Elution behaviour of the enzyme on a phosphocellulose column suggests that glucose induces a specific phosphate site on the enzyme to which the phosphate bearing regulatory ligands of the enzyme may bind.     

The complete amino acid sequence of the catalytic domain of rat brain hexokinase, deduced from the cloned cDNA

The complete amino acid sequence of the catalytic domain of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been deduced from the nucleotide sequence of cloned cDNA. Extensive similarity in sequence, taken to indicate similarity in secondary and tertiary structure, is seen between the mammalian enzyme and yeast hexokinase isozymes A and B. All residues critical for binding glucose to the yeast enzyme are conserved in brain hexokinase. A location for the substrate ATP binding site is proposed based on relation of structural features in the yeast enzyme to characteristics commonly observed in other nucleotide binding enzymes; sequences in regions proposed to be important for binding of ATP to the yeast enzyme are highly conserved in brain hexokinase.     

Purification and properties of rat brain hexokinase

Rat brain hexokinase has been purified to homogeneity as judged by disc-gel electrophoresis, isoelectric focusing, and analytical ultracentrifugation. More than 50% of the initial activity could be obtained in homogeneous form (sp act, 60 units/mg protein) by a simple procedure consisting essentially of two steps: relatively specific solubilization of the enzyme from the mitochondrial membrane by glucose-6-P, followed by DEAE-cellulose column chromatography. The molecular weight is approximately 98,000; this same molecular weight was observed when the denatured enzyme was examined by the SDS-polyacrylamide electrophoretic technique, strongly suggesting that the enzyme consists of a single polypeptide chain. In accord with this view, a single N-terminal amino acid, glycine, has been recovered in 80% yield based on a molecular weight of 98,000. The amino acid composition of the rat brain hexokinase has been determined and found to be very similar to that previously reported for the bovine brain enzyme (Schwartz, G. P., and Basford, R. E. (1967) Biochemistry6, 1070, suggesting extensive sequence homology. A notable feature of the brain hexokinases is a relatively low aromatic amino acid content, as judged by the amino acid composition and the relatively low molar extinction coefficient.     

Effect of inorganic phosphate on the reverse reaction of bovine brain hexokinase

Kinetic studies were used to investigate the mode of brain hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) regulation by glucose 6-phosphate (glucose-6-P), ADP, and inorganic phosphate (Pi). A model for regulation of brain hexokinase by glucose-6-P and Pi had been proposed from initial-rate studies and binding experiments [Ellison, W. R., Lueck, J. D., & Fromm, H. J. (1975) J. Biol. Chem. 250, 1864-1871]. The results of the present investigation demonstrate that Pi is an activator of the brain hexokinase reaction when the reaction is studied in the nonphysiological direction. Evidence is presented which indicates that the back-reaction substrates and Pi can bind the enzyme simultaneously, and the suggestion is made that Pi binds to an allosteric site on the enzyme. These findings are in marked contrast to results obtained in the absence of ADP which convincingly demonstrate that glucose-6-P and Pi are mutually exclusive binding ligands for brain hexokinase. The kinetic data can be reconciled with the model for hexokinase regulation within the context of the well-established kinetic mechanism for brain hexokinase.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

Tubulin and microtubule are potential targets for brain hexokinase binding.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.     

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

Predominance of the Cytoplasmic Form of Brain Hexokinase in Cultured Astrocytes

Abstract: Astrocytes have been cultured from neonatal rat brain according to the flask culture procedure of Booher and Sensenbrenner. Approximately 80% of the hexokinase (ATP: d -hexose 6-phosphotransferase, EC 2.7.1.1) activity is found in the soluble fraction in homogenates of these cells, in contrast to only 20% of the total activity in the soluble fraction of whole brain homogenates. The hexokinase from the cultured astrocytes has been compared with the cytoplasmic and glucose-6-P-solubilized mitochondrial enzymes from whole brain. In kinetic properties and pH-activity relationships, the glial hexokinase was similar to the cytoplasmic enzyme but different from the mitochondrial enzyme of whole brain. Using immunohistochemical methods for detecting hexokinase localization at the electron microscopic level, most of the cells showed prominent staining of cytoplasmic areas. If the cultured astrocytes are accepted as valid models for astrocytes in situ , these results support the suggestion of Bigl and co-workers that the predominant form of hexokinase in glial cells is the cytoplasmic enzyme.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

A large-scale purification of recombinant histone H1.5 from Escherichia coli

The conversion of glucose into glucose 6-phosphate (Glc 6-P)1 traps glucose in a chemical state in which it cannot leave the cell and hence commits glucose to metabolism. In human tissues there are at least three hexokinase isoenzymes responsible for hexose phosphorylation. These enzymes are constituted by a single polypeptide chain with a molecular weight of approximately 100 kDa. Among these isoenzymes, hexokinase type I is the most widely expressed in mammalian tissues and shows reversion of Glc 6-P inhibition by physiological levels of inorganic phosphate. In this work the hexokinase I from human brain was overexpressed in Escherichia coli, as a hexahistidine-tagged protein with the tag extending the C-terminal end. An average of 900 U per liter of culture was obtained. The expressed protein was one-step purified by metal chelate affinity chromatography performed in NTA-agarose column charged with Ni(2+) ions. In order to stabilize the enzymatic activity 0.5 M ammonium sulfate was added to elution buffer. The specific activity of purified hexokinase I was 67.8 U/mg. The recombinant enzyme shows kinetic properties in agreement with those described for the native enzyme, and thus it can be used for biophysical and biochemical investigation.     

An intact hydrophobic N-terminal sequence is critical for binding of rat brain hexokinase to mitochondria

The N-terminal sequence of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) has been determined to be X-NH-Met-Ile-(Ala, Gln)-Ala-Leu-Leu-Ala-Tyr-, where X is a blocking group on the N-terminal methionine, probably an N-acetyl group. Modification of this hydrophobic N-terminal segment by endogenous proteases in crude brain extracts resulted in loss of the ability to bind to mitochondria, but had no effect on catalytic activity, resulting in the appearance of nonbindable enzyme reported by several previous investigators to be present in purified hexokinase preparations. Similar results can be obtained by deliberate limited digestion with chymotrypsin (cleavage points marked by arrows in sequence above). Both bindable and nonbindable enzyme, the latter generated either by endogenous proteases or with chymotrypsin, have an identical C-terminal dipeptide sequence, Ile-Ala. The great susceptibility of the N-terminus to proteolysis plus the marked effect that its proteolytic modification has on binding of hexokinase to anion exchange or hydrophobic (phenyl-Sepharose) matrices suggest that this N-terminal segment is prominently displayed at the enzyme surface. Epitopes recognized by two monoclonal antibodies which block binding of hexokinase to mitochondria (but have no effect on catalytic activity) have been mapped to a 10K fragment cleaved from the N-terminus by limited tryptic digestion. Thus the binding of hexokinase to mitochondria appears to occur via a "binding domain" constituting the N-terminal region of the molecule, with maintenance of an intact hydrophobic sequence at the extreme N-terminus being critical to this interaction. A resulting specific orientation of the molecule on the mitochondrial surface is considered to be a prerequisite for the observed coupling of hexokinase activity and mitochondrial oxidative phosphorylation.     

Glucose phosphorylation in tumor cells. Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K.K., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D.M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a lambda gt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in the rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.     

Glucose catabolism in brain. Intracellular localization of hexokinase

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.     

Rat brain hexokinase: location of the substrate hexose binding site in a structural domain at the C-terminus of the enzyme

A glucose analog, N-(bromoacetyl)-D-glucosamine (GlcNBrAc), previously used to label the glucose binding sites of rat muscle Type II and bovine brain Type I hexokinases, also inactivates rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) with pseudo-first-order kinetics. Inactivation occurs predominantly via a "specific" pathway involving formation of a complex between hexokinase and GlcNBrAc, but significant nonspecific (i.e., without prior complex formation) inactivation also occurs, and equations to describe this behavior are derived. Inactivation is dependent on deprotonation of a residue with an alkaline pKa, consistent with the modified residue being a sulfhydryl group as reported to be the case with the hexokinase of bovine brain. The affinity label modifies three residues (per molecule of enzyme) at indistinguishable rates, but only one of these residues appears to be critical for activity. Amino acid analysis of the modified enzyme indicates derivatization of three cysteine residues; there was no indication of modification of other residues potentially reactive with haloacetyl derivatives. Kinetic analysis and effects of protective ligands were consistent with location of the critical sulfhydryl at the glucose binding site. Peptide mapping techniques permitted localization of the critical residue, and thus the glucose binding site, in a 40-kDa domain at the C-terminus of the enzyme. This is the same domain recently shown to include the ATP binding site. Thus, catalytic function is assigned to the C-terminal domain of rat brain hexokinase.     

用定位突变方法对人脑己糖激酶活性位点的研究

哺乳动物己糖激酶Ⅰ的分子量是１００ｋＤ．目前已经认为是由分子量５０ｋＤ酵母型己糖激酶通过基因复制和融合进化来的．己糖激酶Ⅰ的Ｃ端半分子包含了底物葡萄糖的结合位点即催化位点．Ｘ射线衍射结构的结果已经推测在酵母型的己糖激酶分子中Ｓｅｒ－１５８、Ａｓｐ－２１１是和葡萄糖的结合及催化活性有关,这些氨基酸残基相当于人脑己糖激酶Ⅰ分子中的Ｓｅｒ－６０３、Ａｓｐ－６５７,它们正好位于该酶分子的Ｃ端半分子中．定位突变这两个氨基酸残基得到４个该酶的Ｃ端半分子酶（ｍｉｎｉ－ＨＫⅠ）的突变体,它们是Ｓｅｒ－６０３→Ｃｙｓ,Ｓｅｒ－６０３→Ｔｈｒ,Ａｓｐ－６７５→Ｇｌｕ,Ａｓｐ－６７５→Ｖａｌ．实验结果指出４个突变体酶的Ｋｍ值变化不大,但酶活性只保留野生型酶的０．２８％～１１％,园二色谱分析４个突变体的ＣＤ谱与野生型酶基本一致,因此说明二级结构没有变化．这些研究结果和Ｘ射线衍射结构的推断是一致的,显示了Ｓｅｒ－６０３和Ａｓｐ－６５７氨基酸残基在该酶结合底物葡萄糖或催化作用上起了重要的作用．     

Comparative studies on glucose phosphorylating isoenzymes from vertebrates--VIII. Immunochemical studies on mammalian hexokinases A

An immune serum elicited in a rabbit by injection of homogeneous brain hexokinase A was shown to be specific for the antigen. Other rat hexokinase isoenzymes (hexokinases B, C or D) did not present cross-reaction when tested by immunoinhibition of enzyme activity, double immunodiffusion and immunoadsorbent columns. The enzyme activity of hexokinase A from several mammals (rodents, lagomorphs, artiodactyls) was partially inhibited by the immune serum. In the case of mouse enzyme, the amount of serum required to inhibit 50% of the activity was five-fold higher than in the case of the rat enzyme. Enzymes from cow or sheep brain were only marginally affected. Hexokinases A isolated from various mammals, tested against the rat enzyme, showed faint lines of precipitation and marked spurs in double immunodiffusion plates even when enzymes from closely related rodents were analyzed. Immunoadsorbent columns, on the other hand, were able to retain most of the activity of hexokinases A from the mammals studied. Micro-complement fixation tests showed that hexokinases A from mammals outside the Order Rodentia were only partially recognized by the anti-hexokinase Arat serum. The results suggest that amino acid substitutions on the hexokinase A molecule have occurred at a rather fast rate.     

Functional characteristics of hexokinase bound to the type a and type B sites of bovine brain mitochondria.

Hexokinase is released from Type A sites of brain mitochondria in the presence of glucose 6-phosphate (Glc-6-P); enzyme bound to Type B sites remains bound. Hexokinase of freshly isolated bovine brain mitochondria (Type A:Type B, approximately 40:60) selectively uses intramitochondrial ATP as substrate and is relatively insensitive to the competitive (vs ATP) inhibitor and Glc-6-P analog, 1,5-anhydroglucitol 6-phosphate (1,5-AnG-6-P). After removal of hexokinase bound at Type A sites, the remaining enzyme, bound at Type B sites, does not show selectivity for intramitochondrial ATP and has increased sensitivity to 1,5-AnG-6-P. Thus, the properties of the enzyme bound at Type B sites are modified by removal of hexokinase bound at Type A sites. It is suggested that mechanisms for regulation of mitochondrial hexokinase activity, and thereby cerebral glycolytic metabolism, may depend on the ratio of Type A:Type B sites, which varies in different species.