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PubMed1991

Molecular genetic basis of maple syrup urine disease in a family with two defective alleles for branched chain acyltransferase and localization of the gene to human chromosome 1.

Maple syrup urine disease in humans results from inherited defects in branched chain alpha-ketoacid dehydrogenase, a mitochondrial multienzyme complex. A variety of genetic changes may produce this phenotype by affecting the function of any of the three complex-specific subunits. The varied clinical expression observed in patients may be partially explained by the defects in the involved subunit. Here we report localization of the gene for the branched chain acyltransferase component of the complex to human chromosome 1 and describe a proband who is a compound heterozygote at this locus. One allele, inherited from the father, produces transcripts with 124 nucleotides deleted from the coding region. The deletion is not found in the branched chain acyltransferase gene, implying that the deleted transcripts arise by an error in transcript processing. Cells from the patient's mother contain 50% of the normal amount of mRNA for the subunit, and the proband has inherited this nonexpressing allele from her. As a result, the proband produces no acyltransferase protein and therefore has greatly impaired complex activity. A phenotypically normal sibling is shown to be genetically similar to the mother having inherited the mother's nonexpressing allele and the father's normal allele.

Acyltransferases↗
PubMed1990

Gene-enzyme relationship in the sulfate assimilation pathway of Saccharomyces cerevisiae. Study of the 3'-phosphoadenylylsulfate reductase structural gene.

In yeast, mutations in six different loci (MET1, MET4, MET8, MET16, MET22, and MET25) have been reported to result in the absence of 3'-phosphoadenylylsulfate (PAPS) reductase activity. In the present study, we show that MET16 is the structural gene for PAPS reductase and that the yeast and the Escherichia coli enzymes display significant similarities. Thioredoxin has been implicated in the reduction of PAPS in Saccharomyces cerevisiae as well as in E. coli. One of the generally accepted mechanisms for the action of thioredoxin as a hydrogen donor involves a redox-active sulfhydryl group in the catalytic site of PAPS reductase. However, the present study shows that the site-directed mutagenesis of the unique cysteine from PAPS reductase leads to an enzyme which remains active in vivo. This result would rather support the hypothesis of thioredoxin playing the role of a thiol carrier in the reduction of PAPS into sulfite. Strains separately mutated in the six different loci cited above were examined for the expression of different genes. A mutation in the MET4 gene abolishes transcription of both genes MET16 and MET25. In contrast, mutations in MET1, MET8, and MET25 do not impair MET16 transcription, yet strains bearing these mutations are devoid of PAPS reductase activity. To account for the latter result, we postulate that the enzymes involved in sulfate assimilation may occur as a multienzyme complex in S. cerevisiae.

Amino Acid Sequence↗
PubMed1975

Effects of thyroid hormones on enzymes involved in fatty acid and glycerolipid synthesis.

The influence of thyroid hormones on lipid biosynthesis was studied after administration of L-thyroxine to rats for 5 days. Their weights remained the same as those of control animals, despite an approximately 3-fold increment in plasma L-thyroxine and L-triiodothyronine concentrations. The activity of acetyl-CoA carboxylase and fatty acid synthetase as well as incorporation of tritium into fatty acids were depressed significantly in epididymal adipose tissue and enhanced significantly in livers of thyroxine-treated rats. Using antibodies specific against rat liver fatty acid synthetase, it was determined that the changes in activity of this multienzymic complex were due to alterations in amount of enzyme protein. In the presence of optimal concentrations of fatty acids, radioactive sn-glycero-3-phosphate, and co-substrates, total glycerolipid synthesis (defined in this study as the sum of newly formed radioactive mono- and diacyl-sn-glycero-3-phosphate, diglyceride, and triglyceride) was decreased significantly in adipose tissue and increased in liver and heart. Thus, administration of thyroid hormone results in tissue-specific alterations in lipid biosynthesis which, at least in the case of fatty acid synthetase, are due to changes in enzyme protein content.

Acetyl-CoA Carboxylase↗
PubMed1990

The purification and characterization of arginase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.

Arginase↗
PubMed1987

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells.

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.

2',5'-Oligoadenylate Synthetase↗
PubMed1989

Azelaic acid: mode of action at cellular and subcellular levels.

Azelaic acid (AZA) has been reported to have an inhibitory effect on DNA synthesis of melanoma cell lines. In order to elucidate the mechanism(s) underlying this inhibitory effect, I elected to study the effects of AZA and, for control purposes, adipic acid (ADA) on DNA synthesis rate of nuclei isolated from melanoma cells and keratinocytes cultured in the presence of different concentrations of the dicarboxylic acids. Before doing so, I found, by autoradiography, that [3H]AZA is incorporated into the nuclei in a time-dependent manner. AZA, and to a lesser extent ADA, caused a dose-dependent inhibition of DNA synthesis, regardless of whether these substances were present in cell cultures before isolation of nuclei, or were incubated with already isolated nuclei. In searching for the target for this inhibitory effect on nuclear DNA synthesis, I found that AZA, and to a lesser extent ADA, is a potent inhibitor of both bacterial DNA polymerase and of multienzyme complexes isolated from cultured melanoma cells and keratinocytes. These data suggest that the inhibitory effect of the dicarboxylic acids AZA and ADA on DNA synthesis of several cell lines is due to the interference of these substances with the activation of enzymes (e.g. DNA polymerases) required for DNA synthesis.

Adipates↗
PubMed1989

Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase.

A cDNA clone encoding rat liver aspartyl-tRNA synthetase was isolated by probing a lambda gt11 recombinant cDNA expression library with antibodies directed against the corresponding polypeptide from sheep liver. The 1930-base pairs-long cDNA insert allowed the expression in Escherichia coli of an active enzyme of mammalian origin. The nucleotide sequence of that cDNA, corresponding to the DRS1 gene, was determined. The open reading frame of DRS1 corresponds to a protein of Mr = 57,061, in good agreement with the previously determined molecular weight of the purified enzyme. The deduced amino acid sequence shows extensive homologies with that of yeast cytoplasmic aspartyl-tRNA synthetase, more than 50% of the residues being identical. In rat liver, aspartyl-tRNA synthetase occurs in two distinct forms: a dimeric enzyme and a component of a multienzyme complex comprising the nine aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. The primary structure of the DRS1 gene product is discussed in relation to the occurrence of two distinct forms of that enzyme.

Amino Acid Sequence↗
PubMed1989

cDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase.

Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes. Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery. To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase. The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase. The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus. The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics. Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase. The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.

Amino Acid Sequence↗
PubMedDate not supplied

[Effect of ionizing radiation on the properties of rat liver phosphoprotein phosphatase].

Phosphoproteidphosphatase (3.1.3.16) of high specificity for lysil-tRNA-synthetase (6.1.1.6) and proteins of high-molecular-weight multienzyme complex of aminoacyl-tRNA-synthetases (6.1.1.) was isolated from rat liver. Irradiation of animals with an absolutely lethal dose of 0.21 C/kg decreased phosphoproteidphosphatase activity: a 3-4-fold decrease was noted 1 hr following irradiation. The activity of the enzyme isolated 24 hr after irradiation increased but did not reach the control level.

Acute Disease↗
PubMed1985

Influence of supramolecular structure on the enzyme mechanisms of rat liver lysyl-tRNA synthetase-catalyzed reactions. Synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate.

Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.

Adenine Nucleotides↗
PubMed1987

Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis.

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.

Acetyl Coenzyme A↗
PubMed1988

Microinjection of deoxynucleotides into mouse cells. No evidence that precursors for DNA synthesis are channeled.

The technique of microinjection was applied for the introduction of radioactive, phosphorylated precursors of DNA synthesis into living mouse cells in culture. Autoradiographs proved that DNA was well labeled by injected dTTP, dCTP, dATP, dGTP, and (to a minor extent) dTMP, but the efficiency was much lower when CDP, ADP, or GDP was used. For practical reasons, injections into nuclei were preferred, but injections into cytoplasm showed no principal difference with the autoradiographed nuclei. The kinetics of the uptake of the injected material agreed neatly with the calculation (from pool sizes and polymerization rate) that the intracellular deoxytriphosphates are sufficient for about 10 min of DNA synthesis. All this evidence strongly argues against the concept that precursors of DNA synthesis are channeled in vivo within a multienzyme complex and suggests a free diffusion of deoxynucleotides within the cell. Injected thymidine was less able to enter the deoxy nucleotide metabolism compared with thymidine from the culture medium. A mutant cell line deficient in thymidine kinase did not accumulate intracellular thymidine. These data indicate that thymidine kinase is a membrane associated enzyme and that uptake and phosphorylation of thymidine are coupled reactions.

Animals↗
PubMed1986

Ligand binding-promoted conformational changes in yeast ornithine transcarbamoylase.

It has been proposed that regulatory multienzyme complex formation between yeast ornithine transcarbamoylase (OTCase) and arginase is triggered by a conformational change promoted by the binding of ornithine to a regulatory site in OTCase (Wiame, J.-M. (1971) Curr. Top. Cell. Regul. 4, 1-38). To isolate the binding of ornithine to the proposed regulatory site, the active site was blocked with the high affinity (Ki = 13 +/- 1.4 nM) bisubstrate analogue, delta-N-phosphonacetyl-L-ornithine (PALO). The binding of PALO to the active site produces large changes in the absorption (delta A290-296 = 0.010/mg of enzyme) and in the fluorescence (25% quenching) of the protein. These changes both saturate at one PALO/polypeptide chain. The binding of PALO also changes the rate constant for diffusional acrylamide quenching by 43% and increases the midpoint for the thermal denaturation of the enzyme by 13 degrees C. Finally, PALO binding results in a +2.8% change in the sedimentation coefficient demonstrating that these spectral and energetic changes are associated with a gross structural change in the enzyme. In an effort to detect ligand binding to the proposed effector site on OTCase, ornithine was added to the enzyme saturated with PALO, and consequent conformational changes were tested for using methodologies identical to those which demonstrated active site ligand binding-promoted conformational changes. In no instance were any additional differences observed. Hence, strong support for isosteric effector binding-promoted conformational changes cannot be presented. We conclude that active site ligand binding events themselves are responsible for conformational changes which promote enzyme-enzyme association of OTCase with arginase.

Binding Sites↗
PubMed1978

An active alpha'2beta2 derivative of tryptophean synthase formed by limited proteolysis.

A new approach to studying the arrangement of subunits in the multienzyme complex tryptophan synthase is reported. Comparative studies of limited tryptic proteolysis of the alpha2beta2 complex and of the separate beta2 and alpha subunits show that subunit association inhibits two types of proteolysis which occur with the separate subunits: (i) cleavage of the beta2 subunit to two fragments with consequent loss of activity and (ii) complete degradation of the alpha subunit with loss of activity. Trypsin treatment of the alpha2beta complex does, however, result in at least one cleavage of the alpha subunit and yields an active alpha'2beta2 complex. The alpha'2beta2 complex can be resolved into an active beta2 subunit and an active alpha derivative termed alpha'. These two species can reassociate into the active alpha'2beta2 complex. alpha' derivative can be separated into a large fragment of Mr approximately 20,000 to 23,000 and a small peptide by polyacrylamide gel electrophoresis under denaturing conditions.

Escherichia coli↗
PubMed1987

The interaction of rat liver carbamoyl phosphate synthetase and ornithine transcarbamoylase with inner mitochondrial membranes.

The intramitochondrial localization of the urea cycle enzymes, carbamoyl phosphate synthetase and ornithine transcarbamoylase, has been examined by both in vitro and in situ studies. The following three lines of evidence are presented to establish that significant fractions of the rat liver enzymes are loosely associated with the inner mitochondrial membrane: 1) when the mitochondrion is fractionated, the enzymes partition between the matrix and membrane fractions in the absence of detergent and partition solely to the matrix in the presence of detergent; 2) the purified enzymes associate with purified inner membrane preparations; and, 3) protein A-gold electron microscopic immunocytochemical analysis of rat liver sections reveals a nonrandom arrangement of the enzyme, with the maximal enzyme density adjacent to the inner mitochondrial membrane. These findings serve as the basis for novel potential mechanisms for regulation of the activity of the enzymes and provide additional evidence for the extensive organization of the mitochondrial matrix. The membrane interaction might also serve as the organizing factor for a carbamoyl phosphate synthetase-ornithine transcarbamoylase or other multienzyme complex.

Animals↗
PubMed1984

Mutational analysis of primosome assembly sites. I. Distinct classes of mutants in the pBR322 Escherichia coli factor Y DNA effector sequences.

The assembly of the primosome, a multienzyme complex responsible for priming of lagging-strand DNA synthesis in Escherichia coli, occurs on defined regions of DNA. These primosome assembly sites are on the order of 70 nucleotides in length, yet they share little DNA sequence homology. In order to understand the interaction of the primosomal proteins with these sites, the isolation of single-base substitution mutants of the wild-type sequences has been undertaken. The response of 32 of these mutated primosome assembly sites to increasing concentrations of monovalent and divalent cations when they were used as DNA effectors for E. coli replication factor Y-catalyzed ATP hydrolysis and their efficiency as primosome-dependent DNA replication templates have revealed the existence of four distinct classes of mutations in primosome assembly sites. Class I mutations have essentially no effect on the activities elicited by the DNA site; thus, it is likely that they define nonessential or spacer nucleotide residues. Class II mutated DNAs require higher Mg2+ concentrations than the wild-type DNA to be fully activated as factor Y ATPase effectors and cannot be stimulated in the ATPase reaction by monovalent salt at suboptimal levels of Mg2+. The implication of this mutant phenotype on the role of secondary and tertiary DNA structure in determining an active site is examined in the accompanying article (Soeller, W., Abarzúa, P., and Marians, K. J. (1984) J. Biol. Chem. 259, 14293-14300). Class III mutations coinactivate both the ATPase effector and DNA replication template activity of the site, indicating that they probably represent essential contact points between factor Y and the DNA. Class IV mutated DNAs behave in a manner similar to class II mutated DNAs in the ATPase reaction, but have a replication template activity intermediate between that of the class III and class II mutant DNAs. It is possible that these mutant DNAs are deficient in their ability to catalyze, during primosome assembly, a step subsequent to that of factor Y binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗
PubMed1984

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes.

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.

Animals↗