Syncatalytic sulfhydryl group modification in mitochondrial aspartate aminotransferase from chicken and pig heart

One sulfhydryl group of the mitochondrial isoenzyme of aspartate aminotransferase from both chicken and pig heart exhibits syncatalytic reactivity changes similar to those found previously in the cytosolic isoenzyme from pig heart (Birchmeier, W., Wilson, K.J., and Christen, P. (1973) J. Biol. Chem. $\text{248}$, 1751–1759). The reactivity of the only titratable sulfhydryl group toward 5,5′-dithiobis-(2-nitrobenzoate) is at a minimum in the free pyridoxal and pyridoxamine form of the enzyme and is increased by approximately one order of magnitude when covalent enzyme-substrate intermediates are formed. The modification of the sulfhydryl group does not affect enzymatic activity. This finding supports the earlier conclusion that the syncatalytic reactivity changes are not due to a direct participation of this group in the active site but rather to conformational adaptations of the enzyme-coenzyme-substrate compound occurring in the catalytic mechanism of aspartate aminotransferases.     

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

Conspicuous degree of homology between the mitochondrial aspartate aminotransferases from chicken and pig heart.

The sequence of 40 amino acid residues at the amino terminus of mitochondrial aspartate aminotransferase from chicken heart differs in only 2 positions from the sequence of mitochondrial aminotransferase of pig heart. Close structural similarity had been suggested by previous data on syncatalytic sulfhydryl modifications (Gehring H., and Christen P. (1975) Biochem. Biophys. Res. Commun. $\text{63}$, 441–447). The cytosolic aspartate aminotransferases from the same two species have now been found to differ considerably in the mode of their syncatalytic modifications. The data suggest that the cytosolic and mitochondrial aspartate aminotransferases might have evolved at different organelle-specific rates.     

Mitochondrial and cytosolic aspartate aminotransferase from chicken: Activity toward aromatic amino acids

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase from chicken heart accept as substrates L-phenylalanine, L-tyrosine and L-tryptophan. The specific activities of the mitochondrial isoenzyme toward these substrates are between 0.1 to 0.5% of that toward aspartate and two orders of magnitude higher than that toward alanine. The specific activities of the cytosolic isoenzyme toward the aromatic substrates are 10 to 70% of the respective values of the mitochondrial isoenzyme. The activities of both isoenzymes toward aromatic amino acids are increased two- to threefold by 1 M formate. Larger increases by formate were observed for the alanine aminotransferase activity of both isoenzymes whereas their aspartate aminotransferase activity was inhibited by formate. The opposite effects of formate on the activities toward the aromatic and aliphatic monocarboxylic substrates on the one hand and the dicarboxylic substrate on the other are consonant with the notion of formate occupying the binding site of the distal carboxylate group of the substrate (Morino Y., Osman A.M., and Okamoto M. (1974) J. Biol. Chem. $\text{249}$, 6684–6692). Apparently, in the ternary complex of aspartate aminotransferase with formate and aromatic amino acids, the aromatic rings of the latter bind to a site which does not overlap with the binding site for the distal carboxylate.     

Preferential utilization of glutamine for amination of xanthosine 5'-phosphate to guanosine 5'-phosphate by purified enzymes from Escherichia coli

GMP synthetase was purified 180-fold from $\text{E. coli}$ B and 18-fold from the derepressed purine auxotroph, $\text{E. coli}$ B-96. The enzymes from both sources show the same preference for glutamine over ammonia as amino donor. Each is dimeric, consisting of subunits of molecular weight about 60,000. Thus the two are apparently identical. The similarities between GMP synthetase and xanthosine 5′-phosphate aminase of $\text{E. coli}$ B-96 (N. Sakamoto, G.W. Hatfield, and H.S. Moyed, J. Biol. Chem. (1972) $\text{247}$, 5880–5887) in respect to structure, state of derepression, and behavior during purification, lead us to the conclusion that the synthetase and the aminase are a single entity. We observe no loss or separation of glutamine-dependent activity upon purification of GMP synthetase and we suggest that such loss, reported by other workers, results artifactually by inactivation of an intrinsic glutamine-binding site. GMP synthetase appears not to contain a glutamine-binding subunit which is separable from the xanthosine 5′-phosphate-aminating component.     

The hydrolytic and transacylation activity of the phospholipase A1 purified from human post-heparin plasma.

This study demonstrated that the phospholipase A₁ purified from human post-heparin plasma catalyzes the same reactions (hydrolysis and transacylation) and utilizes the same substrates as the phospholipase A₁ obtained by heparin treatment of the plasmalemma of rat liver (Waite, M. and Sisson, P. (1973) $\text{J. Biol. Chem. 248}$, 7985). 1-acylglycerol was the preferred acyl donor and transacylation was the predominate reaction. The results strongly support our earlier conclusions that the phospholipase in plasma originates from the liver and that this enzyme is capable of using a variety of acyl acceptors, including water.     

Inactivation of glutamate decarboxylase by bromopyruvate

Bromopyruvate was shown to inhibit $\text{E. coli}$ glutamate decarboxylase competitively with respect to L-glutamate. High concentrations of bromopyruvate caused a time-dependent inactivation of glutamate decarboxylase. However, the apoenzyme was rapidly and irreversibly inactivated by bromopyruvate with an inactivation constant of 490 1 mole^?1 min^?1 at pH 5.7. Studies with labeled bromopyruvate indicated that approximately 1.7 moles of inhibitor were bound per subunit of apoenzyme.     

Regulation of coenzyme utilization by the dual nucleotide-specific glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroids.

The dual wavelength assay technique (H. R. Levy, and G. H. Daouk, 1979, J. Biol. Chem.254, 4843–4847) is used to examine the rates of the NADP- and NAD-linked reactions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase simultaneously under various conditions. Inhibition by ATP, MgATP^2?, acetyl-CoA, and palmitoyl-CoA is greatly diminished at high glucose 6-P concentration which favors the NAD-linked reaction. Increasing $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratios inhibit the NADP-linked, but stimulate the NAD-linked reaction. The selective effects of glucose 6-P and the $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ concentration ratio, which cannot be detected by conventional assays, are explained in terms of the differing kinetic mechanisms for the NADP-linked and NAD-linked reactions previously described (C. Olive, M. E. Geroch, and H. R. Levy, 1971, J. Biol. Chem.246, 2047–2057). It is proposed that these effects constitute the mechanism whereby the nucleotide specificity of the amphibolic glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides is regulated.     

The intracellular location of hepatic fructose 1,6-biphosphate aldolase.

Foemmel $\text{et}\text{al.}$ (J. Biol. Chem. $\text{250}$, 1892–1897, 1975) have presented evidence that rat liver aldolase exists $\text{in}\text{situ}$ as a specific complex with the endoplasmic reticulum. In the present study we have examined the alternative possibility that binding of enzyme to the particulate element represents an artifact of the dilution of the ionic constituents of the cytoplasmic milieu. To this end, procedures were developed for homogenization and subfractionation which effected less than a 3-fold dilution of the intracellular content. Using these procedures, virtually all of the liver aldolase was recovered in the soluble supernatant fraction. We conclude that aldolase is not associated with the endoplasmic reticulum $\text{in}\text{situ}$.     

Outer membrane of gram-negative bacteria. XVII. Secificity of transport process catalyzed by the lambda-receptor protein in Escherichia coli.

The outer membrane of Gram-negative bacteria contains (a) “porin” proteins that form transmembrane channels and allow diffusion of various hydrophilic, small molecules (Nakae, J. Biol. Chem., 251, 2176–2178, 1976), and (b) proteins which catalyze the specific transport of unique classes of compounds, e.g. the λ-receptor protein facilitates the diffusion of maltose and maltotriose (Szmelcman et al., Eur. J. Biochem., 65, 13–19, 1976). When strains of $\text{Escherichia coli}$, $\text{B}\text{r}$ and K-12 containing the λ-receptor but not porin were constructed and compared with those containing neither of them, it was found that in the former strains the transmembrane diffusion of glucose and lactose, but not of histidine and 6-aminopenicillanic acid, was significantly accelerated. These results suggest that λ-receptor may facilitate the diffusion of sugars other than maltose.     

Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease

A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.     

Explanation for the apparent inactivation of tyrosine aminotransferase in hepatoma cells by concanavalin A

Brief treatment of hepatoma cells in monolayer culture with concanavalin A causes a decrease in tyrosine aminotransferase specific activity that is thought to be a rapid, reversible inactivation of the enzyme (T.V. Gopalakrishnam and E.B. Thompson 1977 J. Biol. Chem. $\text{252}$, 2717–2725). We confirm this decrease, but attribute it to an increased leakage of cellular protein from concanavalin A-treated monolayer cultures during the harvesting procedure. If the cells are washed free of medium and lysed $\text{in}$,$\text{situ}$ by freezing and thawing them, or by treating them with buffer containing a nonionic detergent, equal amounts of tyrosine aminotransferase are found in concanavalin A-treated and untreated cells. If cells are harvested by scraping them from the substrate, some tyrosine aminotransferase is lost into the buffer used to collect the cells. Treatment of cells with concanavalin A markedly increases the amount of enzyme lost during this procedure, and results in a low enzyme content in the washed cells. No inactivation occurs, however, because the total amount of tyrosine aminotransferase present in the cell pellet and the wash buffer is equal for treated and untreated cells.     

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. $\text{249}$, 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. $\text{8}$, 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m₃², 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.     

On the existence of a nucleotide-binding regulatory site on brain hexokinase.

Binding of ADP to rat brain hexokinase provided protection against inactivation of the enzyme by glutaraldehyde or by chymotryptic digestion. Graphical analysis of the inactivation experiments was, in both cases, consistent with the existence of a single ADP binding site and a K_d ≈ 3mM for the hexokinase-ADP complex. Both Cibacron Blue F3GA and tetraiodofluorescein, previously found to have a general affinity for nucleotide binding sites, were competitive (vs. ATP) inhibitors of the enzyme, suggesting that they bound only to the site occupied by the nucleotide substrate, ATP. While alternate interpretations cannot be excluded, it is felt that these results are most consistent with the view that there is a single nucleotide binding site on the enzyme. They thereby may serve to stimulate a search for alternative explanations for the complex inhibitory pattern of ADP which had previously been attributed to the existence of two ADP binding sites on the enzyme (J. Ning, D.L. Purich, and H.J. Fromm, $\text{J. Biol. Chem. 244}$, 3840–3846 (1969).     

Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.

A cytochrome $\text{c}$ - cytochrome $\text{c}$ oxidase complex containing 0.8–1.0 moles of cytochrome $\text{c}$ per mole of cytochrome $\text{c}$ oxidase (heme a + a₃) was isolated as described by Ferguson-Miller, S., Brautigan, D.L., and Margoliash E., J. Biol. Chem. $\text{251}$, 1104 (1976). This complex was reacted with dithiobissuccinimidyl propionate, an 11 Å bridging bifunctional reagent, and the cross-linked products obtained were analyzed by two dimensional gel electrophoresis. Cytochrome $\text{c}$ was cross-linked to subunit II of cytochrome $\text{c}$ oxidase. Other cross-linked products were formed involving different subunits of cytochrome $\text{c}$ oxidase. These included I+V, II+V, III+V, V+VII, IV+VI and IV+VII. Experiments are also described using N,N′-bis(3-succinimidyloxycarbonylpropyl) tartarate. The major product formed with this 18 Å bridging bifunctional reagent was a pair containing II+VI.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

The occurrence of ethanolamine and galactofuranosyl residues attached to Penicilliumcharlesii cell wall saccharides

$\text{Penicillium}$$\text{charlesii}$ incorporates ³H or ¹⁴C from ³H- or ¹⁴C-labeled ethanolamine into an -alkali soluble, alcohol -insoluble fraction obtained from cell walls. Dansyl ethanolamine was isolated from this alcohol-insoluble fraction following dansylation and hydrolysis. The alcohol-insoluble material was non-dialyzable and contained galactofuranosyl, glucosyl, phosphoryl, amino acyl and variable quantities of uronosyl residues. The lack of detectable quantities of mannosyl residues in this material suggests that the galactofuranosyl-containing cell wall polymer is distinct from the peptidophosphogalactomannan which is obtained from culture filtrates of $\text{P}$. $\text{charlesii}$ (Gander $\text{et}$$\text{al}$., (1974) J. Biol. Chem. $\text{249}$, 2063).     

A study of the sulfhydryl groups of rat brain hexokinase

Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1) is rapidly inactivated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). The inactivation follows monophasic first-order kinetics in either the absence of ligands (k = 0.641 min^?1 at 25 °C) or in the presence of saturating levels of ATP (free or complexed with Mg²⁺) or P₁; the inactivation rate is slightly increased ($\text{k ? 0.7}$ min ^?1) in the presence of ATP or P₁. In contrast, glucose and glucose-6-P markedly decrease the inactivation rate; inactivation in the presence of these ligands is biphasic, with two first-order rates ($\text{k ? 0.5}$ min^?1 and 0.01 min^?1) being distinguishable.The enzyme contains 14 sulfhydryl groups which react with 5,5′-dithiobis-(2-nitrobenzoate); reaction of these groups in the native enzyme is complete after 2 hr at 25 °C, or in approx 5 min with the urea or guanidine-denatured enzyme. In the native enzyme, three classes of sulfhydryl groups are distinguishable and are designated as F-, I-, or S-type based on their fast (k ? 0.7 min^?1), intermediate ($\text{k ? 0.5-0.7}$ min^?1), or slow ($\text{k ? 0.02}$ min^?1 rates of reaction with 5,5′-dithiobis-(2-nitrobenzoate). The correlation of inactivation rates with the rates for reaction of the I-type sulfhydryls indicates that the I-type sulfhydryls include residues necessary for catalytic activity. The F-type residues are clearly not required for activity.The effects of ATP, P₁, glucose, and glucose-6-P on the reactivity of the sulfhydryls have been determined. As in the absence of ligands, S-, I-, and F-type sulfhydryls could be distinguished. In the presence of saturating concentrations of these ligands, the F, I, and S classes of sulfhydryls contained respectively: with ATP, 1, 4, and 7 residues; with P₁, 1, 3, and 7 residues; with glucose, 1, 2, and 5 residues; with glucose-6-P, 1, 2, and 1 residues. Comparison with rate constants for inactivation in the presence of these ligands again indicated that I-type sulfhydryls were particularly important in maintenance of enzyme activity. The present results indicate considerable similarity between the reactivity of the sulfhydryl residues in rat brain hexokinase and the sulfhydryls of the bovine brain enzyme [V. D. Redkar and U. W. Kenkare (1972), J. Biol. Chem., 247, 7576–7584].     

Evidence for an active-center cysteine in the SH-proteinase α-clostripain through use of N-tosyl-L-lysine chloromethyl ketone

The rapid reaction of α-clostripain with tosyl-L-lysine chloromethyl ketone results in a complete loss of activity and in the disappearance of one titratable SH group whereas the number of histidine residues is not affected. Tosyl-L-phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride have no effect on the catalytic activity. From the molar ratio and under the assumption of 1:1 molar interaction, the fully active enzyme has a specific activity of 650–700 $\text{units}\text{mg}$ [twice the value proposed by Porter et al. (J. Biol. Chem. 246 (1971) 7675-7682)]. Partial oxidation makes it experimentally impossible to attain this maximal value.