Biomedical subjects

N M Kredich

Publications and source records attributed to N M Kredich.

At least 37 records · Page 2Linked to original sources

PubMed1990

In vitro interactions of CysB protein with the cysJIH promoter of Salmonella typhimurium: inhibitory effects of sulfide.

The cysteine regulon of Salmonella typhimurium is positively regulated by the CysB protein and an inducer, which can be either O-acetyl-L-serine or N-acetyl-L-serine. In vivo experiments confirmed that sulfide and L-cysteine (supplied as L-cystine) interfere with induction by exogenously supplied O-acetyl-L-serine and also showed the same effects when N-acetyl-L-serine was used as an inducer. In a gel shift assay, purified CysB protein bound specifically to a 278-base-pair DNA fragment containing the S. typhimurium cysJIH promoter region. Binding occurred in the absence of inducer but did not stimulate in vitro transcription initiation, indicating that binding alone is insufficient to cause formation of a transcription initiation complex. Addition of N-acetyl-L-serine or O-acetyl-L-serine was required for transcription initiation and also stimulated binding three- to eightfold. Sulfide inhibited both transcription initiation and binding by interfering with the stimulatory effects of inducer in a competitive manner. These findings indicate that sulfide is an anti-inducer and may explain why full expression of the cysteine regulon requires sulfur limitation. L-Cysteine did not affect in vitro transcription initiation or binding of CysB protein to the cysJIH promoter region. The in vivo effects of L-cysteine may be secondary to its degradation to sulfide by the inducible enzyme cysteine desulfhydrase.

Bacterial Proteins↗

PubMed1989

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase.

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.

Amino Acid Sequence↗

PubMed1989

Characterization of the cysJIH regions of Salmonella typhimurium and Escherichia coli B. DNA sequences of cysI and cysH and a model for the siroheme-Fe4S4 active center of sulfite reductase hemoprotein based on amino acid homology with spinach nitrite reductase.

The hemoprotein component of Salmonella typhimurium sulfite reductase (NADPH) (EC 1.8.1.2) was purified to homogeneity from cysJ266, a mutant strain lacking sulfite reductase flavoprotein. The siroheme- and Fe4S4-containing enzyme was isolated as a monomeric 63-kDa polypeptide and consisted of a mixture of unligated enzyme and a complex with sulfite. Following reduction with 5'-deazaflavin-EDTA and reoxidation, the complex was converted to the uncomplexed, high spin ferri-siroheme state seen previously with Escherichia coli sulfite reductase hemoprotein preparations. The S. typhimurium hemoprotein exhibited catalytic and physical properties identical to the hemoprotein prepared by urea dissociation of E. coli sulfite reductase holoenzyme and was fully competent in reconstituting NADPH-sulfite reductase activity when combined with excess purified sulfite reductase flavoprotein. The DNA sequences of cysI and cysH from S. typhimurium and E. coli B were determined and, together with previously reported data, confirmed the organization of this region as promoter-cysJ-cysI-cysH with all three genes oriented in the same direction from the promoter. Molecular weights deduced for the cysI-encoded sulfite reductase hemoprotein and for the cysH-encoded 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase were approximately 64,000 and 28,000, respectively. Comparison of the deduced amino acid sequence of sulfite reductase hemoprotein with that of spinach nitrite reductase (Back, E., Burkhart, W., Moyer, M., Privalle, L., and Rothstein, S. (1988) Mol. Gen. Genet. 212, 20-26), which also contains siroheme and an Fe4S4 cluster, showed two groups of cysteine-containing sequences with the structures Cys-(X)3-Cys and Cys-(X)5-Cys, which are homologous in the two enzymes and are postulated to provide the ligands of the Fe4S4 cluster in both proteins. From these sequences and from crystallographic (McRee, D. E., Richardson, D. C., Richardson, J. S., and Siegel, L. M. (1986) J. Biol. Chem. 261, 10277-10281) and spectroscopic data in the literature, a model is proposed for the structure of the active center of these two enzymes.

Amino Acid Sequence↗

PubMed1989

Oral dipyridamole increases plasma adenosine levels in human beings.

Plasma adenosine levels in five healthy volunteers for 5 consecutive days showed far less intrasubject than intersubject variation (p less than 0.0001), indicating that plasma adenosine levels are relatively constant during this period. Plasma adenosine levels were then measured in a different group of five healthy subjects for a 5-day control period and during a 5-day course of oral dipyridamole at a dose of 100 mg every 6 hours. Intrasubject comparisons showed that plasma adenosine levels were significantly higher during the 5 days of dipyridamole administration than during the control period (p = 0.017) and that this increase was most significant after 48 hours of drug (p less than 0.001) administration. The average increase was 0.133 mumol/L (60%) with a range of 0.063 to 0.197 mumol/L (37% to 212%) during the last 3 days. A significant positive correlation was noted between plasma adenosine and dipyridamole levels (p = 0.001). We conclude that adenosine levels are relatively stable for an individual and are maximally increased after 2 days of oral dipyridamole.

Adenosine↗

PubMed1989

Molecular characterization of the cysJIH promoters of Salmonella typhimurium and Escherichia coli: regulation by cysB protein and N-acetyl-L-serine.

The cysJIH promoter regions from Salmonella typhimurium LT7 and Escherichia coli B were cloned and sequenced. Primer extension analyses showed that the major in vivo transcription initiation site in S. typhimurium is located 171 nucleotides upstream of the cysJ start codon. Minor start sites were found 8 and 9 nucleotides downstream of the major site. In vivo transcription initiation in E. coli was found to occur at a single site 66 nucleotides upstream of the cysJ start codon. Primer extension studies also indicated that chromosomal cysJIH transcription is stimulated by sulfur limitation and repressed by growth on L-cystine. Paradoxically, in strains carrying plasmids containing the S. typhimurium cysJIH region, the highest levels of primer extension products were found with RNA from cells grown on L-cystine, even though levels of the proteins encoded by cysJ and cysI were normally repressed. In vitro transcription runoff studies with DNA template from the S. typhimurium cysJIH promoter region showed synthesis of a product originating at the major in vivo start site, which was dependent on the presence of purified cysB protein and either O-acetyl-L-serine or N-acetyl-L-serine. N-Acetyl-L-serine was 10- to 30-fold more active than O-acetyl-L-serine as an in vitro inducer of cysJIH transcription.

Acetylcysteine↗

PubMed1988

Methionine adenosyltransferase and S-adenosylmethionine in the developing rat lens.

Methionine adenosyltransferase (MAT) activity, and the concentration of its reaction product, S-adenosylmethionine (AdoMet), were measured in the lenses of rats of different ages; ranging from 1 day of age to over 1 yr. The highest specific activity of MAT was found in the lenses of the one day old rats (sp. ac. 0.327 units/mg-1 protein). After 1 week the specific activity had dropped to 0.067, and by 6 weeks had declined to adult levels (0.02 units/mg-1 protein). AdoMet concentrations were measured by HPLC in perchloric acid extracts. The highest concentration of AdoMet was found in the lenses of day-old rats (48.2 microM), and gradually declined with increasing age, reaching 5.5 microM in the oldest rats. In addition, the specific activity of MAT was found to be higher in the lens epithelium than in the cortex plus nucleus. The specific activity of MAT is almost an order of magnitude higher in the lens epithelial fraction (0.099 units mg-1 protein) than in the combined cortex plus nucleus fraction (0.011 units mg-1 protein).

Aging↗

PubMed1988

Isolation of Salmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids.

We have prepared a library of Salmonella typhimurium genomic fragments cloned in pBR322 and packaged in P22HT capsids. Plasmids carrying 24 of 26 specific genes searched for were isolated by transduction at frequencies of 1 to 344 per 10(6) plasmid transductants. All 11 known genes of the cysteine regulon were isolated from this library, including cysK, which we had previously been unable to clone in a recombinant plasmid with an Escherichia coli host. This library provides a simple and rapid method for isolating most S. typhimurium genes by using S. typhimurium itself as a host and should be particularly useful for cloning genes that might be deleterious to E. coli.

Bacteriophages↗

PubMed1988

DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH.

Nucleotide sequences of the cysK regions of Salmonella typhimurium and Escherichia coli have been determined. A total of 3,812 and 2,595 nucleotides were sequenced from S. typhimurium and E. coli, respectively. Open reading frames of 323 codons were found in both species and were identified as those of cysK by comparison of deduced amino acid sequences with amino- and carboxyl-terminal amino acid analyses of the S. typhimurium cysK gene product O-acetylserine (thiol)-lyase A. The two cysK DNA sequences were 85% identical, and the deduced amino acid sequences were 96% identical. The major transcription initiation sites for cysK were found to be virtually identical in the two organisms, by using primer extension and S1 nuclease protection techniques. The -35 region corresponding to the major transcription start site was TTCCCC in S. typhimurium and TTCCGC in E. coli. The deviation of these sequences from the consensus sequence TTGACA may reflect the fact that cysK is subject to positive control and requires the cysB regulatory protein for expression. Sequences downstream of cysK were found to include ptsH and a portion of ptsI, thus establishing the exact relationship of cysK with these two genes. A 290-codon open reading frame, which may represent the cysZ gene, was identified upstream of cysK.

Amino Acid Sequence↗

PubMed1987

DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli.

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.

Amino Acid Sequence↗

PubMed1987

Purification of the cysB protein from Salmonella typhimurium.

Using an assay based on polypeptide mobility in one- and two-dimensional polyacrylamide electropherograms, we purified the Salmonella typhimurium cysB protein from an Escherichia coli strain carrying plasmid pGBK14, in which the S. typhimurium cysB gene is under transcriptional control of the strong promoter PL from phage lambda. cysB protein constitutes approximately 4% of total soluble protein in such cells and was obtained with good yield at a purity of 85% after precipitation of nucleic acids with streptomycin sulfate, ammonium sulfate fractionation, and hydrophobic chromatography on methyl agarose. Material with 95% purity was obtained with poor yield by ion-exchange chromatography of native protein and with good yield by size exclusion chromatography of denatured protein in sodium dodecyl sulfate. Physicochemical studies of the purified cysB protein show that it is a tetramer of identical 36-kDa subunits with a pI, amino acid composition, amino-terminal sequence, and carboxyl-terminal amino acid content identical to those known from previous studies or deduced from the cysB DNA sequence. Although O-acetyl-L-serine is believed to bind to cysB protein to form a complex that stimulates transcription of genes of the cysteine regulon, we were unable to demonstrate such an interaction using methods that would have detected binding with a Kd of less than 0.1 mM.

Amino Acid Sequence↗

PubMed1986

Purification and properties of rat lens methionine adenosyltransferase.

Methionine adenosyltransferase (MAT) has been partially purified from rat lenses using a combination of ammonium sulfate fractionation and hydrophobic chromatography on phenyl Sepharose columns. The partially purified enzyme resembles purified Type II MAT from non-hepatic tissues. The Km for methionine is 3.0 microM, and the Km for ATP is 80 microM. The enzyme is activated by potassium ions (25-50 mM), and inhibited by higher concentrations of potassium. A divalent cation (magnesium or manganese) is essential for activity. The Vmax with magnesium is about five times higher than with manganese, but the optimal manganese concentration is around 2.0 mM, compared with 10-20 mM for magnesium. The enzyme is active over a broad pH range, with optimal activity between pH 7.0 and 8.0. The enzyme is inhibited by all three of its products, phosphate, pyrophosphate, and S-adenosylmethionine. Individually phosphate and pyrophosphate are weak inhibitors, but in combination they show a marked synergistic inhibitory effect. Tripolyphosphate is also an effective inhibitor. The inhibition of the enzyme by the cataractogenic agent, dimethylsulfoxide, further confirmed the similarity to Type II MAT.

Animals↗

PubMed1986

Cloning and characterization of the cysAMK region of Salmonella typhimurium.

A total of 30 kilobases of DNA comprising the cysAMK region of S. typhimurium was cloned as a series of fragments in phage lambda 1059. The genetic organization of this region was established through studies of gene expression from fragments subcloned in pBR322 and from blot hydridization analyses of restriction sites in chromosomal DNA from multisite deletion strains. The results give a gene order of cysA-cysM-crr-ptsl-ptsH-cysK over a distance of approximately 12 kilobases. cysM and cysA have been cloned and expressed in pBR322; attempts to obtain stable pBR322 derivatives carrying cysK were unsuccessful.

Chromosome Mapping↗

PubMed1985

S-Adenosylmethionine synthetase from human lymphocytes. Purification and characterization.

S-Adenosylmethionine synthetase has been purified to apparent homogeneity from human chronic lymphocytic leukemia cells. Equilibrium sedimentation studies and denaturing polyacrylamide gel electrophoresis indicate that the native enzyme has a molecular weight of 185,000 and a subunit composition of either alpha alpha' beta 2, alpha 2 beta 2, or alpha' 2 beta 2, where alpha, alpha', and beta are polypeptide chains of molecular weight 53,000, 51,000, and 38,000. The alpha and alpha' subunits appear to be the same polypeptide and presumably differ by some kind of post-translational modification. Stoichiometric studies show that the expected products S-adenosylmethionine, pyrophosphate, and orthophosphate are generated in equimolar amounts. The enzyme exhibits linear kinetics with respect to substrate dependency and product inhibition, except for orthophosphate which shows parabolic noncompetitive inhibition with respect to ATP. Initial velocity studies of substrate dependence and product inhibition indicate a steady state mechanism that is ordered Bi Ter with ATP adding before L-methionine and S-adenosylmethionine as the first product released. Pyrophosphate and orthophosphate, however, appear to be released by a random mechanism. Free Mg2+ is an essential activator with a half-maximal effect at 1.0 mM. The Km and Kia for ATP are 31 microM and 84 microM, and the Km for L-methionine is 3.3 microM. The enzyme also has tripolyphosphatase activity which is stimulated by S-adenosylmethionine.

Adenosine Triphosphate↗

PubMed1984

A radioenzymatic assay for plasma adenosine.

A method for analysis of plasma adenosine which combines the principles of radioisotope dilution and enzymatic catalysis is presented. Plasma from venous heparinized blood containing the adenosine deaminase inhibitor 2'-deoxycoformycin is mixed with a small amount of [3H]adenosine and extracted with perchloric acid. Using highly purified enzyme and [gamma-32P]GTP as the phosphate donor, the neutralized extract then serves as substrate for adenosine kinase, and the AMP product is purified by high-performance liquid chromatography. Adenosine concentrations in plasma are linearly proportional to 32P/3H ratios in the enzymatically synthesized AMP and are calculated from a standard curve. The advantages of the method are: ease of sample preparation; sensitivity of 20 nM in as little as 0.3 ml plasma; 20 samples per day can be analyzed by a single operator. Care must be used when obtaining plasma since cellular contamination will affect results. Using this assay, human plasma adenosine levels are 0.121 +/- 0.054 microM for males and 0.101 +/- 0.067 microM for females.

Adenosine↗

PubMed1983

Measurements of S-adenosylmethionine and L-homocysteine metabolism in cultured human lymphoid cells.

The intracellular content and turnover of S-adenosyl-L-methionine (AdoMet) were measured in cultured human lymphoid cells. AdoMet levels were found to be 59 nmol/ml cell volume in exponentially growing WI-L2 lymphoblasts, 3.3-8.1 nmol/ml cell volume in unstimulated peripheral blood mononuclear cells, and 25-33 nmol/ml cell volume 24-48 h after the latter were stimulated with phytohemagglutinin. Increases in the AdoMet content of stimulated cells occurred within 2 h after addition of lectin. First order, pool turnover rates were of the same order of magnitude (0.029-0.091/min) for all three types of cultured cells, but owing to the differences in AdoMet content, absolute utilization rates differed markedly and were 4.5, 0.12-0.40, and 1.41-1.57 nmol/min/ml cell volume in WI-L2, unstimulated peripheral mononuclear cells, and lectin-stimulated peripheral mononuclear cells, respectively. Measurements of homocysteine accumulation in growth medium and of transsulfuration to cysteine indicate that a minimum of 82% of the AdoMet synthesized by WI-L2 is used for transmethylation. Remethylation of homocysteine by these cells could not be detected. AdoMet synthesis accounts for 20-23% of methionine utilization by WI-L2. Judging from the accumulation of homocysteine in the medium of phytohemagglutinin-stimulated peripheral mononuclear cells, a minimum of 38% of AdoMet synthesized must be used for transmethylation. Even though AdoMet utilization by unstimulated peripheral mononuclear cells is relatively small compared to that of stimulated cells and WI-L2, our data indicate that AdoMet turnover in such "resting" cells is three to five times that estimated for nonhepatic tissues. These findings may be relevant to the hypothesis that lymphoid cells are unusually sensitive to inhibition of transmethylation reactions.

Cells, Cultured↗

PubMed1983

Cloning and physical mapping of the cysB region of Salmonella typhimurium.

The cysB region of Salmonella typhimurium was cloned in pBR322 and localized to a 1.75-kilobase HincII fragment. Two-dimensional protein electropherograms showed levels of the cysB polypeptide chain that were several fold higher in plasmid-bearing strains than in the wild type. Fully derepressed levels of sulfite reductase and O-acetylserine sulfhydrylase in cysB plasmid-bearing strains were only 25% higher than in the wild type, suggesting that the product of this regulatory gene ordinarily is not a limiting factor in the expression of the cysteine regulon. The mapping of cysB deletions by Southern blots showed a good correlation between the genetic and the physical maps of this gene. The supX gene was initially cloned with cysB and is within 0.7 kilobase of cysB.

Bacterial Proteins↗

PubMed1983

S-adenosylhomocysteine catabolism and basis for acquired resistance during treatment of T-cell acute lymphoblastic leukemia with 2'-deoxycoformycin alone and in combination with 9-beta-D-arabinofuranosyladenine.

A patient with refractory T-cell acute lymphoblastic leukemia was treated with eight courses of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), over a 5-month period. After developing resistance to dCF, he responded to treatment with the combination of dCF and 9-beta-D-arabinofuranosyladenine (ara-A). We monitored the levels in plasma and urine of adenosine, 2'-deoxyadenosine, and ara-A as well as the accumulation of their nucleotide derivatives in erythrocytes and circulating lymphoblasts. We also monitored the activities of adenosine deaminase and S-adenosylhomocysteine (AdoHcy) hydrolase and the concentrations of AdoHcy and S-adenosylmethionine in lymphoblasts. Production of 2'-deoxyadenosine was related to both the duration of dCF infusion and the magnitude of cytolysis that occurred during treatment: much more 2'-deoxyadenosine was produced by dCF infusion when disease was active than by the same infusion given during remission. Resistance to dCF was associated with a decrease of greater than 90% in the amount of deoxyadenosine 5'-triphosphate accumulated by circulating lymphoblasts. Infusion of dCF resulted in increases of up to 20-fold in the concentration of AdoHcy in circulating lymphoblasts, causing a decrease in the S-adenosylmethionine:AdoHcy ratio (the "methylation index") from a pretreatment value of greater than 40:1 to less than 4:1. This ratio decreased to 2.5:1 during combined treatment with dCF and ara-A, which caused nearly complete inactivation of lymphoblast AdoHcy hydrolase. Decline in the methylation index was accompanied by inhibition of the methylation of newly synthesized lymphoblast RNA. Impaired ability to catabolize AdoHcy may have contributed to the cytolytic responses to dCF and ara-A, as well as to hepatic and central nervous system toxicity associated with their combined use.

Acute Disease↗

PubMed1982

Identification of the Salmonella typhimurium cysB gene product by two-dimensional protein electrophoresis.

Examination of two-dimensional electropherograms of proteins from wild-type Salmonella typhimurium and 16 different cysB strains permitted the identification of a single 34,500-dalton polypeptide chain with a pI of 7.6 that was the product of cysB. Exclusion chromatography indicated that the native cysB protein is a multimer of at least two and probably four or more such subunits.

Alleles↗