Printed in U.S.A. Kinetic Properties of Hexokinase under Near-physiological Conditions RELATION TO METABOLIC ARREST IN ARTEMIA EMBRYOS DURING ANOXIA*

Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propose that inhibition by glucose-6-P (Kis = 65 microM) occurs primarily by competing with ATP at the active site, resulting in the formation of the dead-end complex, enzyme-glucose-glucose-6-P. Versus glucose, inhibition by glucose-6-P is uncompetitive at pH 8.0 and noncompetitive at pH 6.8. Over a physiologically relevant pH range of 8.0 to 6.8 alterations in Km and Ki values do not account for the reduction in glucose phosphorylation, and no evidence suggests that Artemia hexokinase activity is modulated by reversible binding to intracellular structures. Total aluminum in the embryos is 4.01 +/- 0.36 micrograms/g dry weight, or, based upon tissue hydration, 72 microM. This concentration of aluminum dramatically reduces enzyme activity at pH values less than 7.2, even in the presence of physiological metal ion chelators (citrate, phosphate). When pH, aluminum, citrate, phosphate, substrates, and products were maintained at cellular levels measured under anoxia, we can account for a 90% inhibition of hexokinase relative to activity under control (aerobic) conditions.

Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Ki, = 0.86 mM, Kii = 1.0 mM, K i d = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propose that inhibition by glucose-6-P (& = 6 5 PM) occurs primarily by competing with ATP at the active site, resulting in the formation of the dead-end complex, enzyme-glueose-glucose-6-P. Versus glucose, inhibition by glucose-6-P is uncompetitive at pH 8.0 and noncompetitive at pH 6.8. Over a physiologically relevant pH range of 8.0 to 6.8 alterations in K , and Ki values do not account €or the reduction in glucose phosphorylation, and no evidence suggests that Artemia hexokinase activity is modulated by reversible binding to intracellular structures. Total aluminum in the embryos is 4.01 f 0.36 pg)g dry weight, or, based upon tissue hydration, 72 PM. This concentration of aluminum dramatically reduces enzyme activity at pH values c 7.2, even in the presence of physiological metal ion chelators (citrate, phosphate). When pH, aluminum, citrate, phosphate, substrates, and products were maintained at cellular levels measured under anoxia, we can account for a 90% inhibition of hexokinase relative to activity under control (aerobic) conditions. Hexokinase (EC 2.7.1.1) plays a major role in the regulation of carbohydrate metabolism in mammalian cells that utilize glucose as a primary energy source. Glucose 6-phosphate, a 3 To whom correspondence should be addressed. Tel.: 303-492-6180. product and potent inhibitor of the hexokinase reaction, is presumed to be the primary regulator of activity (Colowick, 1973), although alterations in the subcellular distribution of the enzyme are physiologically important depending on the tissue 1985). It is thought that a negative cross-over point in metabolite concentrations, indicative of enzyme inhibition, is typically not observed at the hexokinase reaction because of product inhibition by glucose-6-P (Rolleston, 1972). However, this scenario is clearly not applicable to embryos of the brine shrimp, Artemia. Biochemical and calorimetric studies have shown that the predominantly trehalose-based metabolism of the developing embryo is brought to a virtual halt upon anaerobic incubation and is quickly reinitiated upon return to aerobic conditions (Ewing and Clegg, 1969;Carpenter and Hand, 1986a;Hand and Gnaiger, 1988). Analyses of glycolytic intermediates in these embryos during aerobic development and anaerobic dormancy have demonstrated that trehalase, hexokinase and phosphofructokinase are inhibited under anaerobic conditions Hand 1986a, 1986b;Hand and Carpenter, 1986). Because the hexokinase inhibition occurs while cellular levels of glucose-6-P are decreasing, other regulatory features in addition to product inhibition must be operative. In the present study, a molecular and kinetic analysis was undertaken with hexokinase purified from post-diapause Artemia embryos to explain the inhibition of this enzyme that occurs in vivo during the transition between active and quiescence metabolic states.
Recent observations indicate that alterations of intracellular pH (pHi) may play a primary role in the regulation of carbohydrate metabolism in Artemia. pHi declines rapidly from values 27.9 during aerobic development to 6.0 during anaerobic incubation, eventually reaching pH 6.3 after several hours (Busa et al., 1982). Artificial acidification of pH, to 6.8 by elevated CO, (aerobic acidosis) results in a suppression of respiration rate (Busa and Crowe, 1983) and a blockage of carbohydrate metabolism characterized by cross-over points essentially identical to those under anoxic conditions (Carpenter and Hand, 1986a). It is appropriate to note that while both the ATP:ADP ratio and adenylate energy charge sharply decline under anoxia, they remain constant under aerobic acidosis (Carpenter and Hand, 1986a). Thus, changes in adenylates are not necessary for the observed hexokinase inhibition. As a consequence, all experiments in the present communication, including characterization of hexokinase reaction mechanism and studies of subcellular distribution and inhibition characteristics, were performed at pH 8.0 and 6.8 in order to detect any pH-dependent changes in these properties.
Finally, Womack and Colowick (1979) reported that aluminum ion at micromolar concentrations can inhibit hexokinase activity in a pH-dependent manner. Until now, studies have not experimentally addressed the potential metabolic significance of this finding, but rather have viewed aluminum only as a common contaminant of ATP preparations. Consid-ering the acute pH transition that occurs during entry into anaerobic dormancy, we felt it germane to measure the aluminum content of Artemia embryos and evaluate the inhibition of hexokinase a t physiological levels of this metal ion and its major cellular chelators. EXPERIMENTAL PROCEDURES' In all cases, concentrations of the varied substrate bracketed the K , value, and assays were typically performed in duplicate. When values deviated by more than lo%, quadruplicate assays were done. The average standard error within a set of assays was 5.1% of the quadruplicate mean. Lineweaver-Burk plots of initial velocity measurements were constructed for diagnostic purposes, but the lines given are those predicted by the appropriate rate equations. The nonlinear least squares regression procedure of the Statistical Analysis System (SAS Institute, Cary, NC) was used to determine kinetic parameters, their standard errors, and goodness of fit characteristics. The equations used to fit the various kinetic models were adopted from Segel(l975). Equation 1 predicts enzyme velocity in a sequential mechanism.
Equation 5 describes partially noncompetitive inhibition. In these equations, u is measured velocity, V, is maximal velocity, Kd and K,B are Michaelis constants, KIA is an apparent dissociation constant, KIS is a slope inhibition constant, and KII and KID are intercept inhibition constants.
The criteria suggested by Mannervik (1983) and Motulsky and Ransnas (1987) were used to determine which models best fit the data. The univariate statistics procedure of the Statistical Analysis System was used to determine the error structure of the data and to test the normality of the residual distribution. There was no significant relationship between variance and any dependent or independent variable. Thus equal weight was assigned to each measurement for the fitting process.

RESULTS
Physical Properties and Substrate Specificity-Hexokinase purified from Artemia embryos has a native molecular weight of 40,000 * 1,500 (S.E., n = 3) based on gel exclusion chromatography with Sephacryl S-200. This M, is consistent with reported values from other invertebrates (Stetten and Goldsmith, 1981;Moser et al., 1980;Mochizuki and Hori, 1977). The isoelectric point of Artenia hexokinase was determined by chromatofocusing to be between pH 4.35 and 4.50, which is somewhat lower than PI values reported for yeast and mammalian hexokinases (Colowick, 1973;Wilson, 1985).
Artemia enzyme displays substantial catalytic capacity with Portions of this paper (including parts of "Experimental Procedures" and "Results," Table I , are presented in miniprint at the end of this paper. The abbreviations used are: DTT, 1,4dithiothreitol; HPLC, high performance liquid chromatography; PMSF, phenylmetbylsulfonyl fluoride. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of this Journal that is available from Waverly Press. a variety of sugars (Table 11), one characteristic which distinguishes it as a hexokinase rather than a glucokinase. The minimal activity with galactose suggests that an axial orientation of the C-4 hydroxyl group may sterically hinder phosphorylation at the nearby C-6 position, as suggested by Sols and Crane (1954) for mammalian type I hexokinase. In contrast, substitution or isomerization at the C-2 position (2deoxy-D-glucose, glucosamine; mannose) does not reduce activity as severely. Di-and trisaccharides were not good substrates. In a separate experiment, enzyme activity was measured in the presence of 3 mM inosine triphosphate and 5 mM glucose and was found to be approximately 10% of the activity with ATP as phosphoryl donor. This level of activity with ITP is comparable to other hexokinases, but is substantially higher than that observed for mammalian glucokinase (Colowick, 1973).
Kinetic Studies with Artemia Hexokinase-The reaction mechanism for Artemia hexokinase was studied by examining patterns of product inhibition at pH 8.0 and 6.8. Results obtained at pH 8.0 are shown in Figs. 1-4 (Miniprint) and are described below. Since the patterns of inhibition observed a t pH 6.8 were generally similar, these results will only be addressed when qualitatively distinct.
Plots of l/velocity uersus ]/[ATP], and l/velocity uersus l/[glucose] at several concentrations of cosubstrate resulted in intersections in the second quadrant. These results support a ternary complex reaction mechanism for Artemia hexokinase. There was no indication of substrate inhibition by either glucose or ATP up to concentrations of 10 and 5 mM, respectively.
At saturating concentrations of cosubstrate, ADP was found to be an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP. In experiments with ATP fixed near its K,, the uncompetitive inhibition uersus glucose becomes noncompetitive. When glucose was held at subsaturating concentrations, the inhibition versus ATP remained partial noncompetitive. The partial noncompetitive inhibition is characterized by a hyperbolic replot of intercepts, and is described by Equation 5. At all four combinations of pH (8.0 and 6.8) and glucose (5 and 0.2 mM), fitting the data to this equation reduced the residual sums of squares compared to the fit provided by the standard description of noncompetitive inhibition (Equation 4). Inhibition by glucose-6-P was somewhat more complex. The pattern was noncompetitive versus glucose at pH 6.8, whereas, inhibition at pH 8.0 was uncompetitive. At high glucose-6-P concentrations (24 mM, Fig. 3), the values depart from those predicted by the uncompetitive model, which suggests a noncompetitive C, competitive; NC, noncompetitive; UC, uncompetitive; pNC, partial noncompetitive (slope-linear, intercept-NC model did not significantly improve the fit. hyperbolic).
" p values represent the probability that the NC model improved the fit over the UC model.     (Busa et al., 1982;Busa and Crowe, 1983). *Values taken from Carpenter and Hand (1986a) after 4 h of aerobic development, or after 4 h of aerobic development followed by 4 h of aerobic acidosis or 4 h of anaerobic dormancy. component at very high levels of inhibitor. Inhibition versus ATP was competitive at concentrations of glucose-6-P less than 1.0 mM, with the inhibition pattern again becoming mixed at higher concentrations. However, this noncompetitive component was seen only at glucose-6-P concentrations 5-20-fold greater than in vivo levels. These data suggest that glucose-6-P inhibits enzyme activity primarily by operating at the ATP site, and only has low affinity for the glucose binding site. The patterns of product inhibition observed for Artemia hexokinase are summarized in Table 111. Analysis of slope and intercept replots, with their associated 95% confi-dence interval, indicated that a linear model adequately describes all data sets except for the intercept-hyperbolic pattern of ADP inhibition versus ATP. The kinetic constants which resulted from the various fits are found in Table IV. Since the K,,, for ATP and the Ki for ADP are near the cellular concentrations of these two metabolites, perturbation of the embryonic adenylate energy charge (Atkinson, 1977) would be expected to influence hexokinase activity. Adenine nucleotides were measured in embryos under conditions of aerobic development, aerobic acidosis, and anaerobic dormancy (Table V). Enzyme activity was assayed at these adenylate levels in the presence of 1 mM glucose and 2 mM MgCL and at applicable pH values. Relative to hexokinase activity at pH 8.0 and energy charge of active embryos (0.83), the enzyme activity is reduced by about 62% at conditions found in dormant anaerobic embryos (pH 6.3; adenylate energy charge, 0.29). Hence, the combination of lowered pH and energy charge encountered during anaerobic dormancy can account for a significant reduction of hexokinase activity. In contrast, enzyme activity under conditions of aerobic acidosis (pH 6.8; adenylate energy charge, 0.81) is only depressed by 28% relative to the aerobic value. Because there is a dramatic inhibition of hexokinase in vivo under aerobic acidosis, we felt that there was likely another effector influencing enzyme activity at low pH.
In addition to glucose-6-P and ADP, several other metabolites were evaluated for potential inhibitory effects of Artemia hexokinase activity. Glucose 1,6-diphosphate is claimed to be a potent inhibitor of hexokinase at low pH (Beitner, 1985); but, at concentrations up to 500 pM, this compound did not alter enzyme activity at pH 8.0, 6.8, or 6.3 in the presence of 1.5 mM ATP and 3 mM MgCL. Fructose, 1,6diphosphate, fructose 2,6-diphosphate, and guanosine diphosphate were also ineffective as inhibitors.
Aluminum Inhibition-Aluminum dramatically inhibited Artemia hexokinase activity at pH 7.0 but not at pH 8.2 (Fig.  5A). From these data, half-maximal inhibition at pH 7.0 can be estimated to occur at about 0.5 p~ aluminum. Until now, this inhibition has been viewed as an artifact of ATP contamination and biologically unimportant. However, the pH dependence and the severity of the inhibition by aluminum prompted us to investigate the physiological significance of the phenomenon in Artemia embryos.
Total aluminum in Artemia embryos was measured to be 4.01 f 0.36 pg/g dry tissue (S.E., n = 11) by graphite furnace atomic absorption spectrophotometry. Based on tissue hydration values, the concentration of aluminum in the embryo is approximately 72 p~. Fig. 5B compares the pH profiles of hexokinase activity in the presence of 72 p M aluminum and in its absence (i.e. 0.5 mM citrate). At a pH value correspond-   Table V, except that glucose in all assays was 1.0 mM. Aluminum (as AIC1,) was 72 PM, and MgClz was 2 mM. Enzyme rate was measured as the number of micromoles of glucose-6-P formed per min in a 1.0-ml reaction mixture over 25 min at 25 "C. Blanks were reaction mixtures with deionized water replacing enzyme. Incubations were stopped by the addition of perchloric acid to 6%. Following neutralization by potassium carbonate, glucose-6-P was measured fluorometrically (Lowry and Passonneau, 1972 ing to aerobic development (8.0), aluminum at this concentration inhibits the enzyme by 13%. Upon lowering the pH below 7.2, 72 pM aluminum depresses enzyme activity by 198%. It is appropriate to note that pH, is 56.8 during quiescent metabolic conditions (Busa et al., 1982). This concentration of aluminum, which reflects the total metal content of the embryo, is presumably reduced in vivo by complexation of AP' by physiological metal ion chelators. Citrate and orthophosphate are the major small-molecule chelators of aluminum in the cell (Martin, 1986), and their concentrations were also measured in embryos (Table V). When included in assay mixtures, both citrate and phosphate partially offset the aluminum inhibition. However, the pH sensitivity of hexokinase activity is still very acute (circles, Fig. 5B). As shown in the inset to Fig. 5B, this inhibition is observed at pH 6.8 at aluminum concentrations as low as 10 pM. In a final experiment, we measured hexokinase activity with the discontinuous assay described in Table VI, which permits including both ADP and glucose-6-P simultaneously. The physiological levels of pH, chelators, and metabolites that were used are given in Table V. MgClz was present at 3 mM and AlCl, at 72 p~. When compared to the activity under conditions simulating aerobic development, hexokinase is inhibited by 88% under aerobic acidosis and 91% under anaerobic dormancy (Table VI). Binding Experiments-Hexokinase activity in crude extracts of Artemia embryos is predominantly in the soluble fraction following centrifugation at 20,000 X g. Incubation of 1000 X g pellets and supernatants with 1 mM glucose-6-P (known to elute type I hexokinase from mitochondria; Chou and Wilson, 1972) did not increase hexokinase activity in the 20,000 X g supernatant. In experiments utilizing sucrose homogenization, the soluble hexokinase still accounted for 290% of the total enzyme activity. This figure does not change as a function of the metabolic state of the embryos (aerobic development or anaerobic dormancy). Experiments with purified enzyme failed to indicate any interaction of the enzyme with isolated mitochondria or yolk platelets. Thus, we have no evidence at present that regulation of Artemia hexokinase activity is dependent on reversible binding to intracellular structures.

DISCUSSION
The primary focus of this study was to examine the catalytic and physical properties of hexokinase from Artemia embryos in order to identify the molecular characteristics responsible for the reduction in flux at this step in glycolysis during metabolic arrest in the species (Carpenter and Hand, 1986a). Product inhibition, reaction mechanism, subcellular distribution, and aluminum inhibition are interpreted in the context of metabolic control in Artemia embryos, and related to hexokinases from other sources. The results have implications for the evolution of hexokinase homologues.
Reaction Mechanism of Artemia Hexokinase-The patterns of product inhibition observed in the present study are not characteristic of typical ordered or random bireactant mechanisms. However, when all the data are considered, they are consistent with a random mechanism with the preferred order of glucose binding first and glucose-6-P dissociating last. A preference for this route of substrate addition and product release has been reported for hexokinases from yeast (Danenberg and Cleland, 1975) and mammals (Ganson and Fromm, 19851, although the extent to which the mechanism is random is currently debated (Gregoriou et al., 1986;Bass and Fromm, 1987). We feel that in Artemia hexokinase, the pathway is effectively ordered at physiological concentrations of substrates and products, but the random mechanism is required to explain some of the inhibition patterns at high levels of products. It is appropriate to note that the equations of Rudolph and Fromm (1971) for a rapid equilibrium random mechanism do not apply to Artemia enzyme. Scheme I sum- marizes our interpretation of product inhibition studies with Artemia hexokinase. Support for the ordered nature of the mechanism is found in the pattern of inhibition by ADP. In the proposed mechanism, ADP is usually the first product released and, as such, is expected to be a noncompetitive inhibitor when either substrate is held at unsaturated concentrations. At saturating concentrations of first substrate (glucose), the predicted pattern of inhibition versus the second substrate (ATP) remains noncompetitive. In contrast, when the second substrate (ATP) is held a t saturating concentrations, the pattern of inhibition uersus the first substrate (glucose) should change to uncompetitive. Our data fulfill these predictions.
The slope-linear, intercept-hyperbolic noncompetitive inhibition of ADP uersus ATP has been previously documented for yeast hexokinase (Kosow and Rose, 1970;Viola et al., 1982). The explanation offered by Kosow and Rose (1970) for this pattern recognizes two sites of ADP inhibition, an effect on product release and a competitive interaction versus ATP at the active site. As inhibitor and substrate concentrations are increased, the interaction becomes predominantly competitive, giving a hyperbolic replot of intercepts. The observation of competitive inhibition of ADP toward ATP suggests that the mechanism is partly random at high concentrations of substrate and product (Viola et al., 1982). Scheme I includes the formation of an enzyme-glucose-ADP dead-end complex to account for the competitive portion of the hyperbolic inhibition.
Competitive inhibition of glucose-6-P versus ATP is not predicted in a typical random mechanism, but can be resolved by the formation of the dead-end complex enzyme-glucoseglucose-6-P (Scheme I). In a random mechanism, a competitive inhibitor of ATP would be expected to be noncompetitive versus glucose (Fromm, 1983), and this pattern is observed in a variety of hexokinases (Purich et al., 1973). Fitting our glucose-6-P versus glucose data to a noncompetitive model reduced the residual sums of squares relative to the uncompetitive model at both pH 8.0 and 6.8. The improvement in the fit was not significant at pH 8.0. Thus, the simpler model (uncompetitive) was accepted as suggested by Mannervik (1983). This uncompetitive inhibition supports the proposal that there is a preferred route of substrate addition for Artemia hexokinase, since a competitive inhibitor of the second substrate in an ordered mechanism would be uncompetitive versus the first substrate (Fromm, 1983). At the lower pH, the switch to noncompetitive inhibition may reflect either an increased flux through the alternative route of substrate addition or a secondary interaction of glucose-6-P at the glucose site.
Considerable debate exists about the nature of glucose-6-P inhibition of mammalian hexokinases. Fromm and colleagues (Bass and Fromm, 1987) hold that inhibition results from competition between glucose-6-P and ATP for the y-phosphate subsite of the active site, while Cornish-Bowden and others (Gregoriou et al., 1986) favor allosteric modification by glucose-6-P. The latter position is supported by structural studies that present evidence for gene duplication-fusion in the evolution of mammalian hexokinases (White and Wilson, 1987;Schwab and Wilson, 1988). Briefly, the argument holds that the ancestral hexokinase was of the order of 50,000 Da; and duplication of the gene coding for this protein, followed by fusion of the gene copies, has resulted in a 100,000-Da molecule. One active site remained catalytically competent, while an allosteric site arose from modification of the alternative active site. While this may be the case in mammalian isozymes, it cannot adequately explain the acute inhibition by glucose-6-P of Artemia enzyme, which has a native molecular weight of 40,000. Consequently, we feel that the inhibition by glucose-6-P is a result of competition with ATP at the active site.
Absence of Hexokinase Interaction with Mitochondria-Another feature of the enzyme which can be rationalized in light of structural differences between mammalian and Artemia hexokinases is the capacity to bind to mitochondria. Mammalian brain hexokinase possesses three discrete domains: an N-terminal10,OOO-Da polypeptide; a middle domain of 50,000 Da; and a 40,000-Da C-terminal segment (White and Wilson, 1987). The N-terminal polypeptide mediates binding to mitochondria, whereas the catalytic site resides within the Cterminal portion (White and Wilson, 1987). The Artemia enzyme is expected to be homologous to the mammalian Cterminal region, thus lacking the peptide responsible for reversible mitochondrial binding.
Influence of Substrates and Metabolites under in Vivo Conditions-The importance of the individual kinetic constants and pH-related alterations thereof in determining enzyme rate in vivo can only be assessed in context of cellular metabolite concentrations. Using an equation for a sequential mechanism that incorporates competitive inhibition by glucose-6-P (Segel, 1975) and kinetic constants from Table IV (and including a 20% reduction in V , at pH 6.8 relative to pH 8.0), we calculated hexokinase activity a t physiological substrate and product concentrations ( Table V). The calculated rate of hexokinase under anaerobic conditions, applying the pH 6.8 kinetic parameters, is 32% lower than the value calculated for aerobic conditions. Hence, while the decrease in glucose-6-P predicts an activation relative to aerobic conditions, a marked decline in ATP concentration results in moderate inhibition of the enzyme. During the transition from aerobic development to aerobic acidosis, however, ATP levels to not change while glucose-6-P declines, and the predicted velocity under these conditions represents an activation of 20% above aerobic conditions. Because the ADP concentration is relatively stable under these treatments, inhibition by this product should be similar in all three cases. Thus, product inhibition alone cannot explain the negative cross-over observed at the hexokinase reaction during aerobic acidosis (Carpenter and Hand, 1986a), and we felt that another effector is likely to be inhibiting enzyme activity at low pH values.
Aluminum Inhibition: Possible Significance for Metabolic Arrest-Aluminum ion, which has been demonstrated to be a powerful inhibitor of yeast and mammalian hexokinases, is likewise a potent effector of Artemia hexokinase at low pH. This inhibition is presumably due to the formation of Al-ATP-which competes with Mg-ATP2-for the nucleotide binding site (Womack and Colowick, 1979;Neet et al., 1982). Because 50% maximum inhibition occurs at about 0.5 FM aluminum (Fig. 5A) and the K , for Mg-ATP2-is 290 pM, the AI-ATP-appears to bind approximately 3 orders of magnitude tighter to the enzyme than does the substrate. Neet et d. (1982) proposed that AI-ATP-binds to the enzyme-glucose complex, inducing a slow conformational change to an enzyme form which is not catalytically competent. While only steady state velocities are reported for Artemia hexokinase, the observation that citrate activation of the inhibited enzyme required 1-2 min prior to establishment of a linear rate supports this suggestion. Although inhibition by aluminum is severe at pH values below neutrality, the effect is not observed above pH 8.0. There are a number of explanations for this lack of inhibition at higher pH values. The effect of increasing solution pH on mononuclear aluminum species causes the free AP+ concentration to drop dramatically as pH rises (Martin, 1986). High pH values foster the formation of polynuclear aluminum species (Akitt et al., 1972), which may be too large to complex with ATP. Finally AI-ATP-has a pK value of 7.6 and is much less effective as a competitor of Mg-ATP2above this pH (Viola et al., 1980).
To our knowledge prior studies have not experimentally addressed aluminum inhibition of hexokinase at in vivo values of pH and metal ion chelators.
At pH values, metabolite, aluminum, and chelator concentrations corresponding to anaerobic dormancy and aerobic acidosis, hexokinase activity in vitro was inhibited by approximately 90% compared to the activity under conditions which stimulate aerobic development. Our measurements of enzyme activity were performed a t 72 I.LM aluminum, which assumes that the total tissue aluminum exists in soluble form. We recognize that aluminum can form complexes with a variety of small molecules as well as some proteins, and thus we included in our assays its major cellular chelators (citrate and phosphate) at concentrations that reflect total tissue content? Aluminum inhibition could also have implications for Alzheimer's disease (Crapper et al., 1976), dialysis encephalopathy syndrome (Alfrey et al., 1976), and Parkinsonism (Garruto et al., 1984), all of which are correlated with elevated aluminum concentrations in brain tissue. In this context, Lai and Blass (1984) documented that micromolar amounts of aluminum inhibit glycolysis in mammalian brain extracts.

708-71 1
In the case of citrate, if the intracellular localization is presumed to be largely mitochondrial, then the cytosolic concentration can be calculated to be much lower than the total tissue content. Assuming a 15-fold citrate concentration gradient between mitochondria and cytosol (Williamson, 1979) and a mitochondrial volume of 5% of the total cellular volume (based upon electron micrographs of Artemia embryos), the cytosolic concentration of citrate is calculated to be 40% lower than the total tissue values. At 72 p~ aluminum and pH 6.8, decreasing the citrate concentration by this percentage in the hexokinase assay resulted in a similar percent reduction in enzymic rate. Similarly, a fraction of the measured phosphate is likely sequestered as insoluble calcium salts. By including total tissue levels of aluminum, citrate, and phosphate, we feel that inhibition that would result from overestimated soluble aluminum is offset to some degree by excessive chelator concentrations. Ultimately, the degree of aluminum inhibition of hexokinase depends on measured values of free metal ion in the cytosol, which are presently unavailable. R e final purificatm step was chromatofocusing on Polybuffer exchanger 94 (1.0 x 20 cn, column: 23 ml/h flow rate) The staning buffer war 25 mM histidinemaOH. pH 6 0. and thc elutmn buffer was a 1 9 dilution of Polybuffer 74 adjusted to pH 4.0 w~t h HCI. Glucose (50 mM) was included in both buffers to help preserve hexokinase activity at low pH. The enryme was dlalyzed extensively agalnct the atanmg buffer before applicauon. Hexokmase rctlvity eluted between pH 4.6 and 4.2, and the enzyme was Immediately titrated to neutrality with NaOH and dialyzed against stabilmtion buffer (35 mM TrnlHCI. pH 8.0. 2 mM DIT. 50 mM glucose). Throughout the puriftcatmn, pratem was measured by the method of Lowry et 81. (1951) as modified by Peterson (1977) the mrrmg enzyme units (Table I). Enzyme purity was 94% as determined by electrophoresx on "on-denaturing gels of 7% acrylamide (Davis, 1964). Gels were stamed for total pratem with fast green dye (Gorovsky et al , 1976) and densltometncally scanned at 630 nm This scheme resulted in a 1200-fold ennchmenent of hexokmase and gave a 9% recovery of concentrated solutions on ice for 1-4 hr prior to initiauon of enzyme assays.
We measured aluminum m Sigma disodium ATP from equine muscle (Lot # SSP-7095) Binding Srudrcs. The subcellular distribution of hexokmase was evaluated in metabdlcally actwe and quiescent embryos. Followmg incubation of embryos under candttions of aerobic development or anaerobic dormancy (Carpenter and Hand. 1986a).
tripllcate samples of embryos were homogenized In 800 mM sucrose. 1 mM DIT. 0.1 mM PMSF and centrifuged at 20.000g for 10 min, a procedure which IS known to preseve enzyme-organelle assmation m mammalian skeletal musclc (Clarke el al.. 1984) and selected mvertebrate ttssues (Plarton and Storey, 1986). The supernatants were combined with 3 volumes of rolub$lrralion buffer (100 mM K-phosphate. pH 7.5.200 mM KCI, I mM DTT. 0. I mM PMSF) and assayed for hexokmase actwty. The pellets were washed t w~e in solubilizatm buffer to liberate any b u n d enzyme.
assessed following the protocol of Adams et al. (1988). The potential interaclmn of hexokinase and Arremio yolk platelets was also investigated. Yolk platelets were purified as Utterback and Hand (1987) and incubated at a conccntntion of 10R/mI for 20 min with enriched enzyme at either pH 8.0 or 6.3 ( S h M Tris, SO mM citrate buffer with ImM and pellet fracoans. MgC12) Following centrifugation (700g). hexokinase actiwty was measured tn supematant Rmding of purified hexokmase to isolated embryo mttochondria (13 mdml protein) was Mearuremenr of merobolires and aluminum. Perchloric amd extracts of freeze-clamped embryos were prepared as in Carpenter and Hand (1986a). Adenine nucleotides were measured with high performance liqutd chromatography using a procedure modified from X 25 cm: 5 p n particle stre) fmm Jones Chromatography and eluted isocratically wlth 0 1 M Sehweinsberg and L w (1980). Nucleotides were separated on an Octadecyl column (4.6 mm potassium phosphate buffer. pH 6.0. T h i s HPLC method is supenor to enzymatically-coupled fluorometric analyses of ADP because it avoids interference by GDP. Citrate content was determined fluorometrically following the method of Lowry and Passonneau (1972). The I 0.016-0.064 unit c~trale lyase m 100 mM TrisRICl buffer (pH 7.6). Inorganic phosphate was ml reaction mixlure contained 10 pM NADH, 8 p M ZnSO4. 1 unit malate dehydrogenase and determmed spectrophotomevically as descnbed by Piper and Love11 (1981). except that 7 5% rather than 2.5% ammonium molybdate was mluded in the reagent.
Aluminum was measured in ATP stocks and Arremia embryos fallowing a procedure madlfied from Crapper et al. (1976). Hydrated embryos were treated with an antiformm completely remove the chorion, followed by extensive nnsing with demnired HtO. solullon (0.4 M NaOH, 60 mM NaZCOZ. 2.5% active hypochlorite) for 30 min on ~c e to Approximately 100 mg of embryos were transfemd to q u a m crucibles. dned at 7WC to Constant dry weight. and ashed at 55WC for 20 h. The ash was resuspended ~n 100 pI 5% HNO? and brought to 1.0 ml with demnized H20. Particulate material which did not dissolve was removed by centrifugation at 1O.ooOg for IO min. AI,+ was measured m the supematant wlth a Varlan Graphite Furnace Atomlc Absorption Spectrophotometer.