Inactivation of rat brain acetylcholinesterase by pyridoxal 5'-phosphate

Effects of pyridoxal 5'-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5'-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5'-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5'-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5'-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5'-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5'-phosphate could be reversed upon exhaustive dialysis of the pyridoxal 5'-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5'-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.     

2',3'-Cyclic nucleotide 3'-phosphohydrolase in the developing chick brain and spinal cord

Developmental changes of the 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in the chick brain and spinal cord are reported. The greater part of increase in enzyme activity occurred between 18 days of incubation and 3 days after hatching in the whole brain, and between 18 and 21 days of incubation in the spinal cord. These periods are those of active myelination in the chick brain and spinal cord, respectively. The possibility was emphasized that 2',3'-cyclic nucleotide 3'-phosphohydrolase can be used as a marker for the myelin sheaths in the developing central nervous system. Comparisons were also made among the developmental changes in the forebrain, midbrain, brain stem, cerebellum, and spinal cord.     

Inactivation of rat brain acetylcholinesterase by pyridoxal 5′-phosphate

Effects of pyridoxal 5′-phosphate on the activity of crude and purified acetylcholinesterase from cerebral hemispheres of adult rat brain were examined. Acetylcholinesterase was completely inactivated by incubation with 0.5 mM pyridoxal 5′-phosphate. The enzyme activity remained unaltered in the presence of analogs of pyridoxal 5′-phosphate, pyridoxal, pyridoxamine and pyridoxamine 5′-phosphate. The inhibition of acetylcholinesterase activity by pyridoxal 5′-phosphate appeared to be of a noncompetitive nature, as determined by Lineweaver-Burk analysis. The inhibitory effect of pyridoxal 5′-phosphate on acetylcholinesterase appeared to be a general one, as the activity of the enzyme from the brains of immature chick and egg-laying hen, and from different tissues of the adult male rats, exhibited a similar pattern in the presence of the inhibitor. The inhibitory effects of pyridoxal 5′-phosphate could be reversed upon exhaustive dialysis of the pyridoxan 5′-phosphate-treated acetylcholinesterase preparations. We propose that the effects of pyridoxal 5′-phosphate are due to its interaction with acetylcholinesterase, and that it can be employed as a useful tool for studying biochemical aspects of this important brain enzyme.     

The inhibition of lysyl oxidase in vivo by isoniazid and its reversal by pyridoxal. Effect on collagen cross-linking in the chick embryo.

Isonicotinic acid hydrazide (isoniazid) causes a large increase in the salt-solubility of collagen when injected into chick embryos; this change is accompanied by the inactivation of lysyl oxidase (EC 1.4.3.13), the enzyme responsible for initiating cross-link formation in collagen and elastin. In addition, isoniazid markedly decreases the liver content of pyridoxal phosphate. The depletion of pyridoxal phosphate takes approx. 6 h, whereas the inhibition of lysyl oxidase and the increase in collagen solubility occur more slowly. A reversal of these effects of isoniazid can be produced by the subsequent injection of a stoichiometric amount of pyridoxal, supporting the role of pyridoxal as a cofactor for lysyl oxidase. Treatment of chick embryos with beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, causes an inhibition of the enzyme, which begins to recover within 24 h but which is not affected by the administration of pyridoxal; with isoniazid inhibition, however, lysyl oxidase activity does not show any sign of recovery by 48 h. It is proposed that isoniazid may cause the inhibition of lysyl oxidase by competing for its obligatory cofactor, pyridoxal phosphate. The potential clinical implications in the therapeutic control of fibrosis are briefly discussed.     

METABOLIC PARAMETERS OF ONTOGENESIS OF ELECTRICAL ACTIVITY IN THE BRAIN. SODIUM-POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE IN DEVELOPING CHICK EMBRYO BRAIN

—(1) The activity of the Na-K ATPase in the particulate fraction of the chick embryo brain has been assayed at different stages of development with the objective of finding whether or not changes in the activity of this enzyme bear any relation to the maturation of spontaneous and evoked electrical activity of the growing chick brain. (2) The specific activity of the enzyme is low on day 6 and it rises rapidly between days 10 and 12, at which time it attains a plateau and remains essentially unchanged from day 12 until day 20. Experimental evidence rules out the possible presence of an inhibitor of the enzyme in 8-day-old brain homogenates, suggesting that these developmental changes in the activity of the enzyme may represent new synthesis of enzyme rather than its activation. The period between days 10 and 12 does not represent a unique stage of general protein synthesis. (3) The chick brain particulate enzyme has an optimum activity at pH 7·4 and at 37°. It is optimally activated by a Na⁺ concentration of 100mm and K⁺ concentration of 20 mm . The enzyme is inhibited by ouabain and Ca²⁺. (4) The results have been discussed.     

Ontogenetic changes in [3H]-spiroperidol binding sites in posthatch chick brain

The ontogenetic development of [3H]-spiroperidol binding sites was measured in the optic tectum, cerebellum, forebrain base, and forebrain roof of 1-, 4-, and 16-day-old chicks. In the chick optic tectum and cerebellum both the density and the total number of [3H]-spiroperidol binding sites increased from 4- to 16-days-posthatch, but no significant differences were found in either brain area across the initial four posthatch days. In the forebrain base, [3H]-spiroperidol receptor density and total binding increased significantly between 1- and 4-days-posthatch, but at 16-days-posthatch there was a slight decrease in receptor density. Binding sites in the forebrain roof were minimal at all ages. As expected, saturation experiments yielded curvilinear plots indicating the presence of high- and low-affinity binding sites. The high-affinity sites probably reflect dopamine D-2 receptors; whereas, the low-affinity sites may reflect other receptor types, possibly serotonin S-2. These results suggest that large doses of haloperidol, which are normally used in chick behavioral research, may produce behavioral effects by antagonizing multiple receptors.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.     

Kinetic studies of the pyridoxal kinase from pig liver: slow-binding inhibition by adenosine tetraphosphopyridoxal

Pyridoxal kinase from pig liver has been purified 10,000-fold to apparent homogeneity. The enzyme is a dimer of subunits of Mr 32,000. The enzyme is strongly inhibited by the product pyridoxal 5'-phosphate. Liver pyridoxamine phosphate oxidase, another enzyme involved in the biosynthesis of pyridoxal 5'-phosphate, is also strongly inhibited by this compound [Wada, H., & Snell, E. E. (1961) J. Biol. Chem. 236, 2089-2095]. Thus, the biosynthesis of pyridoxal 5'-phosphate in the liver might be regulated by the product inhibition of both pyridoxamine phosphate oxidase and pyridoxal kinase. Kinetic studies revealed that the catalytic reaction of liver pyridoxal kinase follows an ordered mechanism in which pyridoxal and ATP bind to the enzyme and ADP and pyridoxal 5'-phosphate are released from the enzyme, in this order. Adenosine tetraphosphopyridoxal was found to be a slow-binding inhibitor of pyridoxal kinase. Pre-steady-state kinetics of the inhibition revealed that the inhibitor and the enzyme form an initial weak complex prior to the formation of a tighter and slowly reversing complex. The overall inhibition constant was 2.4 microM. ATP markedly protects the enzyme against time-dependent inhibition by the inhibitor, whereas another substrate pyridoxal affords no protection. By contrast, adenosine triphosphopyridoxal is not a slow-binding inhibitor of this enzyme.     

Isolation,partial purification,characterization, and tissue distribution of phenylmethylsulfonyl fluoride-inhibited feline carboxypeptidase

Phenylmethylsulfonyl fluoride (PMSF)-inhibited carboxypeptidase from cat liver was purified 148-fold by chromatography on CM- and DEAE-cellulose with 27.3% yield. Molecular weight of the enzyme is 100-110 kD as determined by gel filtration on Sephadex G-150. The enzyme has maximum activity at pH 5.50-5.75; its activity is completely inhibited by PMSF or p-chloromercuribenzoate and partially inhibited by iodoacetamide. EDTA, 2-mercaptoethanol, N-ethylmaleimide, Co²⁺ and Ca²⁺, basic carboxypeptidase inhibitor guanidinoethylmercaptosuccinic acid, and angiotensin-converting enzyme inhibitor captopril do not influence its activity. The enzyme cleaves arginine from enkephalin-Leu5-Arg6 and dansyl-Phe-Leu-Arg to form enkephalin-Leu5 and dansyl-Phe-Leu, respectively, and very slowly cleaves leucine from carbobenzoxy-Gly-Leu. Further cleavage of either enkephalin-Leu5 or dansyl-Phe-Leu was not detected. The highest activity of this enzyme was found in adrenal glands and testicles; this activity was 30% lower in hypophysis, and still lower in liver and kidney. The PMSF-inhibited carboxypeptidase activity in brain was about 6-16 times lower than that in adrenal gland. In brain regions, the highest activity was detected in gray matter of cerebral hemispheres and cerebellum, and slightly lower activity was found in thalamus/hypothalamus, striatum, and hippocampus. The lowest activity was found in quadrigeminal bodies, medulla oblongata, and white matter of cerebral hemispheres. The enzyme exists mainly in soluble form; the activity of membrane-associated enzyme is 7-25% of soluble enzyme activity depending on tissue type. We consider here a possible involvement of PMSF-inhibited carboxypeptidase in the metabolism of biologically active peptides.     

Aspartate aminotransferases ofPanicum miliaceum L. andPanicum antidotale retz.

L-Aspartate: 2-oxoglutarate transaminase was isolated and partially purified from leaves ofPanicum miliaceum (C₄, NAD-malic enzyme type) and ofPanicum antidotale (C₄, NADP-malic enzyme type). In each preparation two isoenzymes with different kinetic properties could be characterized. The enzyme activity was irreversibly inhibited by 2-aminooxyacetic acid and by 2-amino-4-methoxy-3-butenoic acid. The first inhibitor reacted with pyridoxal 5-phosphate, and its inhibition could be reversed by the exchange of the modified coenzyme. The second inhibitor binds not only to the coenzyme pyridoxal 5-phosphate, but also to the apoprotein. The results of the dissociation and reconstitution experiments were in agreement with the kinetic data, showing that the mode of inactivation was different for 2-aminooxyacetic acid and 2-amino-4-methoxy-3-butenoic acid.     

Glutatmine synthetase activity in the chick brain during postnatal growth. Effect of MSO

Glutamine synthetase activity was estimated in the chick cerebral hemispheres, optic lobes and cerebellum between the 1st and the 30th day of postnatal growth. Glutamine synthetase activity is higher in the cerebellum than in the cerebral hemispheres and lowest in the optic lobes at 1 day after hatching; at 30 days after hatching, it is the same in the optic lobes and in the cerebellum and lowest in the cerebral hemispheres. The great increase of glutamine synthetase activity between the 1st and the 4th day after hatching corresponds to the appearance of the heterogeneity of the chick brain glutamate metabolism. The glutamine synthetase activity is inhibited by MSO $\text{in vivo}$ at a concentration of 100 mg kg ^?1 at values of 87, 90 and 89 % in cerebral hemispheres, optic lobes and cerebellum of 1, 2 and 4-day-old chicks. The enzyme inhibition is less pronounced $\text{in vitro}$ and reaches values of about 25 and 75 % for 1 and 10 mM MSO concentrations respectively in the three brain areas of the 1 to 4-day-old chick and values slightly lower in the 30-day-old chick brain.     

EFFECT OF CORTISONE AND THYROXINE ON ACID GLYCOSIDASES IN RAT FOREBRAIN AND CEREBELLUM DURING EARLY POSTNATAL DEVELOPMENT

Abstract— l -Thyroxine (200 μg/100 g B.W.) or cortisone-acetate (5 mg/100 g B.W.), administered for 4 consecutive days, affected several lysosomal enzymes in forebrain and cerebellum of rats during various periods of the first month of postnatal development; their effect was age dependent as well as organ specific. Extending thyroxine or cortisone treatment to 7 days (from day 2 to 9) affected enzyme activity more than the 4-day treatment period.     

Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.     

Passive Avoidance Training Increases Fucokinase Activity in Right Forebrain Base of Day-Old Chicks

Fucokinase (EC 2.7.1.52) activity was estimated in supernatants of homogenate from day-old chick forebrain. Enzyme kinetic studies gave a Km of 4.5 X 10(-6) M and Vmax of 3.72 nmol fucose converted into fucose-1-phosphate/mg prot/h. The pH optimum was 7.5. The enzyme is thus considerably more active than was reported for other species and tissues. There were no differences in enzyme activity between the four forebrain regions studied. One hour after chicks were trained on a one-trial passive avoidance learning paradigm, enzyme activity in the right forebrain base increased 14% over control values (p less than 0.02). The 11.3% increase in activity in the left forebrain base and 10.3% increase in the left roof were not statistically significant. The relationship of this change to the increased fucose incorporation into glycoproteins known to occur over a similar time period and the significance of the lateralization of the increase are discussed.     

The effect of several 2-alkyl- and 4'-O-methylanalogs of pyridoxol on mouse liver pyridoxal kinase activity]

A simple method of isolation of partially purified puridoxal kinase preparation from mouse liver, having specific activity of 600-700 E/mg protein and a 30% yield is described. It is demonstrated that of all number of 2-alkyl- and 4'-O-methyl pyridoxol analogs synthesized, 4'-O-methyl-pyridoxol (Ki=0.2-10(-5) M, Km(pyridoxal)=4-10(-5) M) is the most active competitive inhibitor of pyridoxal kinase. 3-Deoxy-4'-O-methylpyridoxol is a non-competitive inhibitor of pyridoxal kinase, the latter having an affinity for the enzyme 16 times lower than that of 4'-O-methylpyridoxol. 2-Alkyl analogs of pyridoxol exhibit properties of competitive inhibitors; the affinity of 2'-ethylpyridoxol for the enzyme is 5 times lower than that of 2'-methylpyridoxol; corresponding 2-alkyl derivatives of dimethyl ethers of 3-hydroxycinchomeronic acids have no pronounced affinity for the enzyme. The study of the toxic effects of pyridoxol analogs on the central nervous system has revealed inverse dependence between the neurotoxic dose of the compound and its efficiency as an inhibitor of pyridoxal kinase (Km/Ki value).     

Seizure susceptibility in the developing mouse and its relationship to glutamate decarboxylase and pyridoxal phosphate in brain

The relationship between the susceptibility to convulsions, the content of pyridoxal 5′-phosphate and the activity of pyridoxal kinase (EC 2.7.1.35) and glutamate decarboxylase (EC 4.1.1.15) in brain, was studied in the developing mouse. Seizures were induced by pyridoxal phosphate-σ-glutamyl hydrazone (PLPGH), a drug previously reported to reduce the levels of pyridoxal 5′-phosphate and as a consequence to inhibit the activity of glutamate decarboxylase in brain of adult mice. It was found that the seizure pattern, as well as the time of appearance of convulsions, differed between 2- and 5-day old mice and 10-day old or older mice, indicating a progressive increase in seizure susceptibility during development. In brain, pyridoxal kinase activity and pyridoxal 5′-phosphate levels were decreased by the administration of PLPGH at all ages studied, whereas glutamate decarboxylase activity was inhibited less than 25% in 2- and 5-day old mice, and about 50% thereafter. Parallelly, the activation of glutamate decarboxylase by pyridoxal 5′-phosphate added in vitro to control homogenates was less in 2- and 5-day old mice than in older animals. It is concluded that the increase in the susceptibility to seizures induced by PLPGH during development is probably related to the increase observed in the sensitivity of glutamate decarboxylase in vivo to a decrease of pyridoxal 5′-phosphate levels. The correlation between pyridoxal 5′-phosphate, glutamate decarboxylase, and seizure susceptibility seems to be established at about 10 days of age.     

Postnatal ontogeny of uridine kinase in the cerebellum,hypothalamus, and cerebral cortex of the rat

Postnatal developmental patterns of uridine kinase were determined in crude subcellular fractions of the rat cerebellum, hypothalamus and cerebral cortex at ages 3 through 60 days. The highest specific activity and predominant distribution of enzyme was in the 105,000g supernatant of the 3 brain regions. Enzyme activity in hypothalamus and cerebral cortex was maximum at 3 days and decreased with age; in cerebellum it increased through 13 days and decreased thereafter. Thus, the pattern of activity in hypothalamus and cerebral cortex paralleled changes in DNA and RNA synthesis through age 60 days; in cerebellum, it more closely approximated changes in DNA synthesis during early development. Changes inK _m with aging suggest that the brain regions contain more than one form of enzyme. The highest particulate activity was in the microsomal fraction of the cerebellum and hypothalamus at all ages and in the cortex at 35 and 60 days. Relative specific activity for microsomal fractions of the brain regions at 60 days indicate a concentration of the enzyme which may be relevant in the maintenance of RNA activity in adult brain.     

Subtypes of Sodium-Dependent High-Affinity L-[3H]Glutamate Transport Activity: Pharmacologic Specificity and Regulation by Sodium and Potassium

Abstract: Some data suggest that the sodium-dependent, high-affinity L-glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium-dependent transport of L-[³H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by <65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were <10-fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L-Glu, L-aspartate, and L-cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10-fold selective as inhibitors. These included β-N-oxalyl-L-α,β-diaminopropionate, α-methyl-DL-glutamate, (2S, 1′S,2′S)-2-(carboxycyclopropyl)glycine, and (2S, 1′S,2′S,3′S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S, 1′R,2′R)-2-(Carboxycyclopropyl)glycine (L-CCG-II) was identified as the most potent (IC₅₀= 5.5 μM) and selective (60–100-fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L-homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L-CCG-II and L-homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.     

Horse liver alcohol dehydrogenase. A study of the essential lysine residue.

1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.