Extracting synaptic conductances from single membrane potential traces.

In awake animals, the activity of the cerebral cortex is highly complex, with neurons firing irregularly with apparent Poisson statistics. One way to characterize this complexity is to take advantage of the high interconnectivity of cerebral cortex and use intracellular recordings of cortical neurons, which contain information about the activity of thousands of other cortical neurons. Identifying the membrane potential (Vm) to a stochastic process enables the extraction of important statistical signatures of this complex synaptic activity. Typically, one estimates the total synaptic conductances (excitatory and inhibitory) but this type of estimation requires at least two Vm levels and therefore cannot be applied to single Vm traces. We propose here a method to extract excitatory and inhibitory conductances (mean and variance) from single Vm traces. This "VmT method" estimates conductance parameters using maximum likelihood criteria, under the assumption that synaptic conductances are described by gaussian stochastic processes and are integrated by a passive leaky membrane. The method is illustrated using models and is tested on guinea-pig visual cortex neurons in vitro using dynamic-clamp experiments. The VmT method holds promises for extracting conductances from single-trial measurements, which has a high potential for in vivo applications.

The ubiquitous cofactor NADH protects against substrate-induced inhibition of a pyridoxal enzyme.

In the usual reaction catalyzed by D-amino acid transaminase, cleavage of the alpha-H bond is followed by the reversible transfer of the alpha-NH2 to a keto acid cosubstrate in a two-step reaction mediated by the two vitamin B6 forms pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP). We report here a reaction not on the main pathway, i.e., beta-decarboxylation of D-aspartate to D-alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, beta-C-C bond cleavage of the single substrate D-aspartate occurs rather than the usual alpha-bond cleavage in the transaminase reaction. The D-alanine produced from D-aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D-aspartate turnover and D-alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzyme.D-aspartate complex with an apparent dissociation constant of 16 microM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of beta-decarboxylase activity by NADH occurs concomitant with a reduction in the 415-nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. alpha-Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 microM. In addition to its known role in shuttling electrons in oxidation-reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme-substrate intermediates. The D-aspartate-induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.

Catalytic ability and stability of two recombinant mutants of D-amino acid transaminase involved in coenzyme binding.

Of the major amino acid side chains that anchor pyridoxal 5'-phosphate at the coenzyme binding site of bacterial D-amino acid transaminase, two have been substituted using site-directed mutagenesis. Thus, Ser-180 was changed to an Ala (S180A) with little effect on enzyme activity, but replacement of Tyr-31 by Gln (Y31Q) led to 99% loss of activity. Titration of SH groups of the native Y31Q enzyme with DTNB proceeded much faster and to a greater extent than the corresponding titration for the native wild-type and S180A mutant enzymes. The stability of each mutant to denaturing agents such as urea or guanidine was similar, i.e., in their PLP forms, S180A and Y31Q lost 50% of their activities at a 5-15% lower concentration of urea or guanidine than did the wild-type enzyme. Upon removal of denaturing agent, significant activity was restored in the absence of added pyridoxal 5'-phosphate, but addition of thiols was required. In spite of its low activity, Y31Q was able to form the PMP form of the enzyme just as readily as the wild-type and the S180A enzymes in the presence of normal D-amino acid substrates. However, beta-chloro-D-alanine was a much better substrate and inactivator of the Y31Q enzyme than it was for the wild-type or S180A enzymes, most likely because the Y31Q mutant formed the pyridoxamine 5-phosphate form more rapidly than the other two enzymes. The stereochemical fidelity of the Y31Q recombinant mutant enzyme was much less than that of the S180A and wild-type enzymes because racemase activity, i.e., conversion of L-alanine to D-alanine, was higher than for the wild-type or S180A mutant enzymes, perhaps because the coenzyme has more flexibility in this mutant enzyme.

The effect of dietary protein on cystine excretion in patients with cystinuria.

Seven patients with homozygous cystinuria were studied on a metabolic ward to determine the effect of dietary manipulation on urinary cystine excretion. Isocaloric diets were calculated based on body weight and activity. Balance studies were performed for 5 days on a low protein diet (9.4 +/- 1.0% total calories) with equal amounts of animal and plant protein sources and an additional 5 days on a high protein (27 +/- 3.0% of total calories) with 70% animal protein. A significant (p less than 0.01) decrease in the excretion of 1/2-cystine, histidine, 3-methylhistidine, 1-methylhistidine, lysine, and ornithine plus arginine occurred on the low protein diet. The mean excretion of 1/2-cystine on the high protein diet was 6.13 +/- 1.48 mMoles per 24 hours which decreased to 4.89 +/- 1.06 mMoles per 24 hours on the low protein diet (p less than 0.001). Thirty seven percent of urine collections during the study were less than 2 liters/24 hours despite the fact that patients were encouraged to drink enough fluid to make 3 liters of urine/24 hours. For patients whose customary diet is high in animal protein, a more vegetarian diet may significantly reduce urinary cystine excretion. Cystine excretion in patients with cystinuria may be significantly different from day to day if animal protein consumption is variable.

Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins.

An allosteric modulator of oxygen release in human erythrocytes is 2,3-diphosphoglycerate, but bovine erythrocytes apparently utilize chloride for this purpose since they contain little, if any, 2,3-diphosphoglycerate. In order to identify the sites to which these anions bind, the site-specific acetylating agent, methyl acetyl phosphate, has been employed to compete with these allosteric modulators and to mimic their effects on hemoglobin function. With human hemoglobin A, methyl acetyl phosphate competes with 2,3-diphosphoglycerate and acetylates only Val-1(beta), Lys-82(beta), and Lys-144(beta) within or near the cleft that binds this organic phosphate (Ueno, H., Pospischil, M. A., Manning, J. M., and Kluger, R. (1986) Arch Biochem. Biophys. 244, 795). With bovine hemoglobin, the acetylation is competitive with chloride ion. The sites of acetylation in oxy bovine hemoglobin are Met-1(beta) and Lys-81(beta) and for deoxy bovine hemoglobin, they are Val-1(alpha) and Lys-81(beta). Thus, these sites are expected to be involved in the binding of chloride to bovine hemoglobin. Treatment of either human or bovine hemoglobins with methyl acetyl phosphate under anaerobic conditions leads to a lowering of their oxygen affinity and hence the covalent modifier has the same effect on hemoglobin function as the non-covalent regulators, 2,3-diphosphoglycerate and chloride. The Hill's coefficient of hemoglobin is unaffected by treatment with methyl acetyl phosphate. Under aerobic conditions, specifically acetylated bovine hemoglobin also has a lowered oxygen affinity, and human hemoglobin A shows a slight change in its oxygen affinity. In general, bovine hemoglobin is more responsive than human hemoglobin to both chloride and methyl acetyl phosphate; the latter agent results in a permanent covalent labeling of the protein. Therefore, the results support the idea that methyl acetyl phosphate may be a useful probe for deciphering the sites of binding of anions to proteins.

Inhibition of the gelation of extracellular and intracellular hemoglobin S by selective acetylation with methyl acetyl phosphate.

Methyl acetyl phosphate binds to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin and selectively acetylates three amino groups at or near that site. The subsequent binding of 2,3-DPG is thus impeded. When intact sickle cells are exposed to methyl acetyl phosphate, their abnormally high density under anaerobic conditions is reduced to the density range of oxygenated, nonsickling erythrocytes. This change is probably due to a combination of direct and indirect effects induced by the specific acetylation. The direct effect is on the solubility of deoxyhemoglobin S, which is increased from 17 g/dL for unmodified hemoglobin S to 22 g/dL for acetylated hemoglobin S at pH 6.8. Acetylated hemoglobin S does not gel at pH 7.4, up to a concentration of 32 g/dL. The indirect effect could be due to the decreased binding of 2,3-DPG to deoxyhemoglobin S within the sickle erythrocyte, thus hindering the conversion of oxyhemoglobin S to the gelling form, deoxyhemoglobin S.

Methyl acetyl phosphate: a novel acetylating agent. Its site-specific modification of human hemoglobin A.

A novel acetylating agent, methyl acetyl phosphate (MAP), has been designed to react with a nucleophile near an anion binding site of proteins. We examined the effect of MAP on hemoglobin (Hb), which has a well defined binding site for 2,3-diphosphoglycerate (DPG), to determine whether this reagent recognizes the DPG binding site. The progress of the reaction was monitored by ion-exchange high-performance liquid chromatography (HPLC) on a TSK CM-SW column. Modified Hb was initially chromatographed on CM-52 and then separated into its component chains. The alpha- and beta-chains from modified and unmodified Hb were digested by TPCK-trypsin. The peptide mixtures were chromatographed on Whatman ODS-3 reversed-phase HPLC columns and the peptide maps of modified and unmodified chains were compared. The peaks formed by the modification with MAP were further purified on YMC ODS-S5 columns and then subjected to amino acid analysis on a Dionex D-500 instrument after acid hydrolysis. We found that the newly formed peptides are beta T1 and beta T14 + 15 and that the loss of a peptide corresponding to beta T9 and beta T 10 + 11 is significant. No change in the alpha-chains was observed. The results suggest that MAP is indeed specific for the DPG binding site, as the above peptides contain the amino acid residues involved in the binding of DPG. We have assigned the acetylation sites as Val-1(beta), Lys-82(beta) and Lys-144(beta).

Site-specific modification of hemoglobin by methyl acetyl phosphate.

Methyl acetyl phosphate, which was originally synthesized as a site-specific reagent for hydroxybutyrate dehydrogenase [R. Kluger and W.-C. Tsui (1980) J. Org. Chem. 45, 2723], also has an affinity for the binding site for 2,3-diphosphoglycerate in hemoglobin. Three residues in or near this cleft between the beta-chains are acetylated by this reagent, i.e., Val-1, Lys-82, and Lys-144. There is no detectable acetylation of any of the amino groups of the alpha-chain. These results indicate the specificity of methyl acetyl phosphate in its reaction with hemoglobin.

Inactivation of nitric oxide synthase after prolonged incubation of mouse macrophages with IFN-gamma and bacterial lipopolysaccharide.

Large amounts of nitric oxide (NO) are produced by the inducible isoform of NO synthase (iNOS) in many cell types once the iNOS gene is transcriptionally activated. In primary mouse peritoneal macrophages elicited by thioglycolate broth, expression of iNOS follows treatment with IFN-gamma and is synergistically increased by the addition of bacterial LPS. Expression of iNOS is suppressible at transcriptional and translational levels by certain cytokines and microbial products. The present study describes a novel form of inactivation of iNOS that is post-translational and nondegradative. Mouse peritoneal macrophages cultured in the presence of IFN-gamma alone or IFN-gamma plus LPS rapidly depleted the medium of L-arginine, a substrate for iNOS, and stopped producing NO. Repletion of L-arginine permitted cells treated with IFN-gamma alone to resume NO production for at least 5 days, leading to the release of more NO than macrophages were previously believed capable of generating. L-Arginine repletion also boosted NO production by macrophages cultured for up to 2 to 3 days in the presence of IFN-gamma plus LPS, but thereafter, iNOS was inactive in these cells whether or not L-arginine was repleted. Activity of iNOS could be restored by adding both L-arginine and fresh IFN-gamma with or without LPS, likely reflecting the synthesis of new enzyme. However, the inactivation of iNOS seen late in culture with a single application of IFN-gamma plus LPS could be attributed neither to loss of iNOS protein nor to its autoinactivation by NO. Thus, LPS, a co-inducer of iNOS, causes macrophages to inactivate iNOS about 3 days after the onset of its induction. The mechanism, which remains to be identified, is novel for iNOS, in that it decreases neither its amount nor its apparent molecular mass.

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme.

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.

Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.

Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.

In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild-type enzyme but at attenuated rates, may yield information on the factors controlling the stereochemistry of the reaction as well as on the catalytic steps of the transaminase pathway.

Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines.

During protein biosynthesis, processing of the N terminus of many proteins may occur through acetylation and deacetylation. The enzyme acylpeptide hydrolase is likely involved in deacetylation of nascent peptide chains or of bioactive peptides. The related enzyme, acylase, hydrolyzes the acetyl amino acid product of the acylpeptide hydrolase reaction to acetate and a free amino acid. There is a reciprocal relationship between the substrates for these enzymes (i.e., substrates for one enzyme are competitive inhibitors for the other). In several cultured cell lines, including normal and malignant cells, the ratio of acylpeptide hydrolase to acylase enzyme activities appears to be coordinated and characteristic for a given cell type. Thus, in normal cultured lung cells, hamster ovary cells, hepatoma cells, and lymphocyte cells, nearly equal amounts of these enzymes are expressed, conducive to optimal processing of acetylated N-terminal residues. Four lines of erythroleukemic cell lines were found to express nearly twice as much acylase as acylpeptide hydrolase activity. In the Ehrlich ascites tumor cell line, where 80% of the proteins have been reported to remain acetylated at their N terminus, acylpeptide hydrolase is hardly expressed but acylase activity is not reduced. The 3p21 region of human chromosome 3, which contains the DNF15S2 locus that encodes acylpeptide hydrolase (Jones et al., Proc Natl Acad Sci USA 1991;88:2194), undergoes deletion in some carcinoma cells; the gene that encodes for the acylase is also present on region 3p of the same chromosome. We found that both acylpeptide hydrolase and acylase activities are practically absent in six small-cell lung carcinoma cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)