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.     

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.     

Inactivation of aspartate aminotransferase during transamination with chloropyruvate. Evidence against modification of Cys390 in cytosolic isoenzyme.

Both the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from pig heart were inactivated during transamination with chloropyruvate. Inactivation occurred with L-alanine as the amino group donor in the presence of potassium formate. When L-glutamate or L-aspartate was employed as the amino group donor in the transamination reaction with chloropyruvate, no inactivation occurred. This is in contrast to the case of inactivation by bromopyruvate (Okamoto, M. &; Morino, Y. (1973) J. Biol. Chem. $\text{248}$, 82–90) where these natural dicarboxylic amino acid substrates were effective in the transamination reaction leading to syncatalytic inactivation (Birchmeier, W. &; Christen, P. (1974) J. Biol. Chem. $\text{249}$, 6311–6315). The Cys₃₉₀ in the cytosolic isoenzyme which was modified in the syncatalytic inactivation was not modified under the present condition for inactivation with either chloropyruvate or bromopyruvate.     

beta-Sulfur substituted alpha-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes

The RS-isomers of beta-mercapto-alpha-ketoglutarate, beta-methylmercapto-alpha-ketoglutarate and beta-methylmercapto-alpha-hydroxyglutarate have been synthesized. Beta-Mercapto-alpha-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart (Ki = 5 nM; Km (DL-isocitrate)/Ki(RS-beta-mercapto-alpha-ketoglutarate) = 650) and pig liver, the cytosolic isozyme from pig liver (I0.5 = 23 nM), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. beta-Mercapto-alpha-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. beta-Mercapto-alpha-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. beta-Methylmercapto-alpha-ketoglutarate (I0.5 = 10 microM) was a much less effective inhibitor than the beta-mercapto derivative for heart NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. beta-Methylmercapto-alpha-hydroxyglutarate, the expected product of reduction of beta-methylmercapto-alpha-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The beta-sulfur substituted alpha-ketoglutarate derivatives were alternate substrates for alpha-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.     

Sulphydryl groups involved in Na+-Li+ exchange in human erythrocytes

Oxidative stress causes cellular injury that is thought to be due to increased cytosolic cation levels. Disturbances of a variety of mechanisms which normally maintain intracellular anion/cation homeostasis, occur during oxidative stress. Reactivity of the SH- groups essential for oubain-resistant Na(+)-Li(+) exchange by N-ethylmaleimide (NEM) and selenite was studied in human erythrocytes. In addition, the reactivity of the substances on SH- groups and Li(+) influx have been studied as a function of pH of the medium. The results show that NEM induces an irreversible inhibition of Li(+) influx. It diminishes progressively with the increasing pH of the medium. Whereas we obtain increasing intracellular Li(+) concentration with the rising selenite concentration in the medium. The maximum effect with this substance is reached at about pH 8.0. We can state that the -SH reagents (NEM and selenite) studied behave differently: NEM inhibits Li(+) influx by modifying the essential SH-groups of the membrane proteins in such a way that the exchange is reduced, whereas it maintains the Na(+) permeability almost unaltered. The slight increase in intracellular Na(+) induced by selenite suggests that the oxidative changes in the intracellular sulphydryl groups may constitute an important mechanism for the regulation of the intracellular cations.     

The mitochondrial ATPase. Evidence for a single essential tyrosine residue.

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with p-mercuribenzene sulphonate (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with N-ethylmaleimide to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal pK_a value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.     

Genetic basis for tissue isozymes of glutamine synthetase in elasmobranchs

Tissue-specific isozymes of glutamine synthetase are present in elasmobranchs. A larger isozyme occurs in tissues in which the enzyme is localized in mitochondria (liver, kidney) whereas a smaller form occurs in tissues in which it is cytosolic (brain, spleen, etc.). The nucleotide sequence of spiny dogfish shark (Squalus acanthias) liver glutamine synthetase mRNA, derived from its cDNA, shows there are two in-frame initiation codons (AUG) at the N-terminus which will account for the size differences between the two isozymes. Initiation at the up-stream and down-stream sites would yield peptides of 45,406 and 41,869 mol. wts. representing the precursor of the mitochondrial isozyme and the cytosolic isozyme, respectively. The additional N-terminal 29 amino acids present in the mitochondrial isozyme precursor contains two putative cleavage sites based on the Arg-X-(Phe,Ile,Leu) motif. The predicted two-step processing would remove 14 of the 29 N-terminal amino acids. These 14 amino acids can be predicted to form a very strong amphipathic mitochondrial targeting signal. Their removal would yield a mature peptide of 43,680 mol. wt. The calculated mol. wts. based on the derived amino acid sequence are therefore in good agreement with previous estimates of an approximately 1.5–2-kDa difference between the M_rs of the mitochondrial and cytosolic isozymes. A model for the evolution of the mitochondrial targeting of glutamine synthetase in vertebrates is proposed. Correspondence to: J.W. CampbellThe nucleotide sequence reported will appear in GenBank under accession number U04617     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

In vitro synthesis of precursor forms of pig heart aspartate aminotransferase isozymes

Precursor forms of the isozymes of aspartate aminotransferase from pig heart were synthesized in vitro and purified by binding to specific antibodies. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis showed that the precursor of the cytosolic enzyme has a similar molecular weight to that of the mature protein whereas the precursor of the mitochondrial isozyme has a molecular weight greater than that of the corresponding mature protein (ΔMW ? 2500). Preliminary sequence studies seem to suggest that the precursor of the mitochondrial isozyme has an extra N-terminal peptide sequence while that of the cytosolic protein has only an extra N-terminal methionine residue.     

Subcellular localization of the F1 and F2 isozymes of horse liver aldehyde dehydrogenase

The subcellular distribution and relative amounts of the two isozymes, F1 and F2, of aldehyde dehydrogenase (EC 1.2.1.3) which were recently purified to homogeneity from horse liver (Eckfeldt, J., et al. (1976) J. Biol. Chem.251, 236–240) have been investigated. A fresh horse liver homogenate was fractionated on DEAE-cellulose. The results indicate that approximately 60% of the total pH 7.0 acetaldehyde dehydrogenase activity is due to the F1 isozyme and 40% is due to the F2 isozyme. Several horse livers were then fractionated into subcellular components using a differential centrifugation method. Based on the disulfiram (Antabuse) inhibition and the aldehyde concentration dependence of the enzymatic activity, it appears that the disulfiram-sensitive F1 isozyme (K_m acetaldehyde ? 70 μm) is primarily cytosolic and the disulfiram-insensitive F2 isozyme (K_{m acetaldehyde} ? 0.2 μm) is primarily mitochondrial. Fluorescence studies showed that the acetaldehyde dehydrogenase of the intact mitochondria could utilize only the endogenous pyridine nucleotide pool and not externally added NAD. Also, the ethanol dehydrogenase activity was found to be nearly 10 times the total acetaldehyde dehydrogenase activity when assaying a horse liver homogenate at pH 7.0 and with saturating substrates. The significant differences between this work and the results reported in rat liver are discussed with respect to the physiological importance of the cytosolic and mitochondrial aldehyde dehydrogenase during the ethanol oxidation in vivo.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Primary structure of mitochondrial glutamic oxaloacetic transaminase from rat liver: Comparison with that of the pig heart isozyme

The complete amino acid sequence of the mitochondrial glutamic oxaloacetic transaminase isozyme from rat liver is presented. The sequence contained 401 amino acid residues, 10 of which are methionine. Cyanogen bromide cleavage of mitochondrial glutamic oxaloacetic transaminase produced 12 peptides, one of which contained an internal homoserine residue resulting from incomplete cleavage by cyanogen bromide. The calculated molecular weight was 44,358. The sequence showed 94% homology with that of the corresponding isozyme from pig heart. These findings support the conclusion that the rate of evolution of the mitochondrial isozymes is lower than that of their cytosolic isozymes.     

Exposed sulphydryl groups of chicken haemoglobins: globin localization and effect of oxygenation on their reactivity

The number and the reactivity of the sulphydryl groups of the two major haemoglobin fractions of adult fowl erythrocytes, Hb-1 and Hb-2², have been determined with paramereuribenzoate and iodoacetamide. The number of sulphydryl groups of Hb-1 that react with paramereuribenzoate and their kinetics of combination are dependent on the ligand state of the molecule. Experiments with [¹⁴C]iodoacetamide show that two sulphydryl groups of the β chains are always reactive, though with different kinetics. One sulphydryl group appears reactive only with paramereuribenzoate and only when the molecule is oxygenated.The number of reactive sulphydryl groups of Hb-2 does not change with the ligand state of the molecule but the kinetics of combination is slower for the deoxy form. Reaction with [¹⁴C]iodoaoetamide shows that each α chain has one fast-reacting sulphydryl group and eachβ chain has one fast and one slowreacting sulphydryl group. The fast-reacting groups of Hb-2 can be blocked selectively with iodoacetamide. Tentative identification of the reacting sulphydryl groups of Hb-2 has been made on the basis of their corresponding positions in human haemoglobin.     

Purification and characterization of the isozymes of phosphoenolpyruvate carboxykinase from rabbit liver

Procedures are described for the purification of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase from rabbit liver. Examination of the purified isozymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated apparent homogeneity and identical molecular weights of approximately 65,000. Gel filtration chromatography of the native isozymes, however, yielded apparent molecular weights of 68,000 and 56,000 for the cytosolic and mitochondrial isozymes, respectively. The isoelectric points as determined by chromatofocusing were 5.8 for the mitochondrial isozyme and 5.0 for the cytosolic isozyme. The purified isozymes were readily separable on ion-exchange columns, with the cytosolic isozyme showing the greater affinity. A minor amount of cross-reactivity was apparent when each isozyme was immunotitrated with polyclonal antibodies raised in goat against the opposite isozyme. Peptide maps obtained by high pressure liquid chromatography of both tryptic digests and cyanogen bromide digests of the isozymes showed that many of the peaks were not coincident, suggesting that differences in the sequences are found throughout the primary structures of the isozymes.     

Synthesis of glutamic oxaloacetic transaminase isozymes in rat liver cells

The synthesis of glutamic oxaloacetic transaminase isozymes in rat liver explants was studied using specific antisera against the cytosolic and mitochondrial isozymes. The pulse-labeled cytosolic isozyme was detected in the cytosolic fraction and remained there in pulse-chase experiments. On the other hand, the pulse-labeled mitochondrial isozyme was detected as a larger precursor in the cytosolic fraction. During chase, the amount of pulse-labeled precursor of the mitochondrial isozyme decreased and labeled mature mitochondrial isozyme appeared in the mitochondrial fraction.     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Inhibition of actomyosin ATPase by high concentrations of 5-hydroxytryptamine. Possible basis of lesion in 5HT-induced experimental myopathy.

The effect of 5-hydroxytryptamine (5HT) on the ATPase activity and sulphydryl group reactivity of mammalian skeletal muscle actomyosin has been studied. 5HT inhibited the Mg2+-activated but not the Ca2+-activated ATPase activity of actomyosin. It slightly activated myosin ATPase. The sulphydryl groups of actomyosin reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) were blocked by concentrations of 5HT which inhibited the Mg2+-activated ATPase. The significance of the results are discussed in relation to the muscle lesions in the experimental myopathy induced by 5HT and imipramine.     

Continuous measurement of mitochondrial pH gradients in isolated hepatocytes by difference ratio spectroscopy

The pH gradient, delta pH, present across the inner mitochondrial membrane in isolated rat hepatocytes was continuously monitored with a novel spectroscopic technique that utilizes the weak acid fluorescein. Unlike most cytosolic pH indicators, such as 2',7'-bis(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF), fluorescein freely distributes between the cytosolic and mitochondrial compartments. As is typical for weak acids, the distribution between these two compartments is governed by the magnitude of the pH gradient. Since fluorescein has two ionizable groups, the fluorescein dianion is concentrated in the mitochondrial compartment 100-fold per delta pH unit. In this compartment, fluorescein absorbance (or excitation) spectra are red-shifted about 6-8 nm in the matrix environment, as compared to the cytosolic dye at equivalent pH values. The combination of favorable mitochondrial accumulation and red-shifted spectra enables mitochondrial pH to be continuously monitored qualitatively in whole cells by dual wavelength spectroscopy (510 minus 540 nm). When the cytosolic pH is determined by independent means, the mitochondrial pH can be quantitated, based on the theoretical dependence of the fluorescein distribution ratio on delta pH, the ratio of cytosolic to mitochondrial volumes, and the known extinction coefficients for the dye in the cytosolic and mitochondrial compartments. The sensitivity of the method for following kinetic responses in mitochondrial pH is especially noteworthy; a 0.1-unit change in delta pH is easily distinguished, with a time resolution of less than a second.     

The solubilization of cytoskeletons of human erythrocyte membranes by p-mercuribenzene sulphonate

The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with $\text{p-}\text{mercuribenzene sulphonate}$ (PMBS) has been followed by measurements of turbidity and the binding of ²⁰³Hg-labelled PMBS. After pretreatment with $\text{N-}\text{ethylmaleimide}$ to block readily reactive sulphydryl groups, incubation with [²⁰³Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups.