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Glutamate elicits several different responses on neurons of isolated ganglia of Aplysia, the most common of which is a hyperpolarization due to conductance increases to either chloride or potassium. We have investigated the actions of aspartate and cysteate on the responses to glutamate. Neither aspartate nor cysteate is potent in activation of glutamate receptors. However both aspartate and cysteate cause a dramatic increase in the response to glutamate when ionophoretically applied before the glutamate application. This potentiating effect of aspartate and cysteate is a result of competition with glutamate for the glutamate transport system, since the potentiation is blocked by cooling and by perfusion with sodium-free sea water. Blockade of glutamate re-uptake by perfusion of sodium-free sea water also causes a significant increase in the response to ionophoretically applied glutamate, which in some neurons may be very large. These results demonstrate that the glutamate reuptake system has an important role in regulation of the responses to glutamate which is similar to that of acetylcholinesterase in regulation of responses to acetylcholine. These observations may be of particular importance in mammalian systems where excess glutamate is associated with neuronal excitotoxicity and cell death.

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Cellular cholesteryl ester metabolism is regulated largely by the balance between intracellular esterification catalyzed by acyl-CoA:cholesterol acyltransferase and cholesteryl ester hydrolysis catalyzed by the cholesteryl ester hydrolases. The hydrolases include both cytosolic and membrane-associated activities; acidic and neutral activities have been described in both compartments. Esterification via the acyltransferase is membrane-associated. Neither the acyltransferase nor the membrane-associated hydrolases have been purified and characterized, and little is known about their genes. Thus, nothing is known about their sizes or structures. Radiation inactivation was used to determine the functional sizes in situ of acyl-coenzyme A:cholesterol acyltransferase, fatty acyl-CoA hydrolase, and acidic and neutral membrane-associated cholesteryl ester hydrolase activities. The functional M(r) +/- SD of the acyltransferase was 213 +/- 35 kD; for the acidic membrane-associated hydrolase, 48 +/- 2 kD; for the neutral membrane-associated hydrolase, 94 +/- 15 kD; and for the fatty acyl-CoA hydrolase, 65 +/- 15 kD. Monoexponential curves were observed in all cases using radiation exposures that inactivated enzyme activities to < or = 10% of control values. Substrate specificity and inhibition studies suggested that the active sites of the acyltransferase and fatty acyl-CoA hydrolase were different, supporting the concept that the hydrolase is not part of the functional unit required for cholesterol esterification.

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Radiation inactivation analysis was utilized to estimate the sizes of the units catalyzing the various activities of hepatic microsomal glucose-6-phosphatase. This technique revealed that the target molecular weights for mannose-6-P phosphohydrolase, glucose-6-P phosphohydrolase, and carbamyl-P:glucose phosphotransferase activities were all about Mr 75,000. These results are consistent with the widely held view that all of these activities are catalyzed by the same protein or proteins. Certain observations indicate that the molecular organization of microsomal glucose-6-phosphatase is better described by the conformational hypothesis which envisions the enzyme as a single covalent structure rather than by the substrate transport model which requires the participation of several physically separate polypeptides. These include the findings: 1) that the target sizes for glucose-6-P phosphohydrolase and carbamyl-P:glucose phosphotransferase activities were not larger than that for mannose-6-P phosphohydrolase in intact microsomes and 2) that the target size for glucose-6-P phosphohydrolase in disrupted microsomes was not less than that observed in intact microsomes. These findings are most consistent with a model for glucose-6-phosphatase of a single polypeptide or a disulfide-linked dimer which spans the endoplasmic reticulum with the various activities of this multifunctional enzyme residing in distinct protein domains.

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Radiation inactivation analysis of liver pieces yielded a target size of 210 kDa for hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase [S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) from rats fed a normal diet. Feeding a diet containing mevinolin and colestipol, which causes a marked increase in enzyme activity, resulted in a reduction of the target size to 120 kDa. These results are consistent with those obtained by radiation inactivation and immunoblotting analysis of isolated microsomes and suggest that the increase in HMG-CoA reductase activity caused by these dietary agents is accompanied by a change from a dimer to a monomer form of the enzyme.

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In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The apparent target size of the polymerase activity of DNA polymerase I in the presence of free radical scavengers was about 40% of that observed in the absence of these agents. This is considerably less than the minimum polypeptide size and may reflect the actual size of the polymerase functional domain. Similar effects, but of lesser magnitude, were observed for glucose-6-phosphate dehydrogenase, nitrate reductase, and sulfite oxidase. These results suggest that secondary damage due to free radicals generated in the local environment as a result of ionizing radiation can influence the apparent target size obtained by this method.

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Sulfite oxidase (EC 1.8.3.1), purified from chicken liver, is comprised of two identical subunits of 55 kDa, each of which contains a molybdenum and heme prosthetic group. The functional size of sulfite oxidase was determined by radiation inactivation analysis using both full, sulfite:cytochrome c reductase, and partial, sulfite:ferricyanide reductase, catalytic activities. Inactivation of full enzyme activity indicated a target size of 42 kDa while the partial activity indicated a target size of 25 kDa. These results confirm the earlier findings of two equivalent subunits and suggest the presence of a functional domain within the subunit structure that contains the molybdenum center and exhibits a smaller molecular mass than that of the enzyme subunit.

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Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.

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The radiation sensitivity of glycoproteins is shown to depend only on the protein portion of the molecule. An artificially created glycoprotein containing glucose-6-phosphate dehydrogenase observes this rule as well as natural enzymes and receptors containing from 2 to 50% carbohydrate. No exceptions have been found. Radiation damage to carbohydrates occurs close to the site of the primary ionization, with little spread of damage into attached polypeptides.

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Assimilatory NADH:nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and Mo6+ per 100-kDa subunit. At low protein concentrations, this tetramer dissociates to a fully active dimer. To further elucidate the possible relationship between quaternary structure and activity, the functional size of nitrate reductase was determined by radiation inactivation analysis at high and low concentrations of enzyme where the principal physical species would be either tetrameric or dimeric, respectively. In both cases, the size obtained by this method was 100 kDa, suggesting that each subunit in the tetramer or dimer can function independently. These results confirm earlier results which indicated that the subunits are identical and that each contains a full complement of prosthetic groups. We also found that the functional sizes of the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase were fractions (approximately 58 kDa, 47 kDa, and 28 kDa, respectively) of the subunit molecular mass, suggesting that these domains are functionally independent.

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Using radiation inactivation and immunoblotting techniques, evidence for functionally active forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase with molecular weights of about 100,000 and 200,000 was obtained. In liver microsomes isolated from rats fed both mevinolin and colestipol, the Mr 100,000 form was the predominant species, whereas in microsomes from animals fed only colestipol, the Mr 200,000 species was the major form. This Mr 200,000 form could be converted to the Mr 100,000 form by addition of dithiothreitol or beta-mercaptoethanol. Although both forms appear to possess catalytic activity, the Mr 200,000 species displays sigmoidal kinetics with respect to the concentration of NADPH, whereas the Mr 100,000 form exhibits typical hyperbolic kinetics.

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Potentiation of the excitatory response to L-glutamate (Glu) by L-aspartate (Asp), similar to that which has been described at the crustacean neuromuscular junction, is observed in Aplysia neurons which are glutamate sensitive. Potentiation of the inhibitory responses to ionophoretically applied Glu in neurons preconditioned with Asp permits experiments which serve to differentiate among four hypotheses previously proposed to explain the underlying mechanism of the phenomenon. The potentiation is inhibited by cooling (Q10 = 1.3 +/- 0.2) and is blocked in Na+-free seawater, where the response to Glu applied alone is increased in both amplitude and duration. These results are most consistent with the view that Glu is normally removed from the extracellular medium through an active reuptake process which is Na+ dependent, is slightly temperature sensitive, and may be blocked by Asp. Potentiation of the excitatory response to L-glutamate (Glu) by L-aspartate (Asp) has been previously described at the crustacean neuromuscular junction (Kravitz et al., 1970; Nistri and Constanti, 1979). This potentiation has been attributed to an Asp-induced change in conformation of the Glu receptor, thereby increasing its affinity for Glu (Shank and Freeman, 1975); suppression of the rate of desensitization of the Glu receptor induced by Asp (Dudel, 1977); blockade by Asp of a Glu reuptake process (Crawford and McBurney, 1977); and release, triggered by Asp, of a bound store of Glu (Constanti and Nistri, 1978).(ABSTRACT TRUNCATED AT 250 WORDS)

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