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J C Rabinowitz

Publications and source records attributed to J C Rabinowitz.

At least 73 records · Page 4Linked to original sources
PubMed1979

Purification of Bacillus subtilis RNA polymerase with heparin-agarose. In vitro transcription of phi 29 DNA.

We have devised a new procedure for the purification of highly active preparations of Bacillus subtilis RNA polymerase holoenzyme. A column of heparin-agarose A-15m is used to rapidly and quantitatively adsorb RNA polymerase from the initial crude extract fraction. This affinity procedure obviates the necessity of including nucleic acid precipitation or partitioning steps and allows for rapid separation of RNA polymerase from proteolytic activity. The enzyme is further purified by preparative glycerol gradient centrifugation resulting in an overall purification in 200-fold in 24 h with near quantitative recovery of polymerase protein and activity. RNA polymerase holoenzyme is obtained by chromatography on single-stranded DNA-agarose. The in vitro transcription products made by purified preparations of B. subtilis and Escherichia coli RNA polymerase holoenzymes in response to B. subtilis phage phi 29 DNA have been analyzed, and an in vitro transcription map is presented. The E. coli RNA polymerase holoenzyme initiates transcription from three promoter sites not efficiently utilized by the B. subtilis holoenzyme under optimal conditions for RNA synthesis.

Bacillus subtilis↗
PubMed1978

Assignment of the cysteinyl 13C nuclear magnetic resonances and comparison of other aliphatic amino acid resonances of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins.

13C NMR spectra of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins show that some 13C resonances of the aliphatic amino acid residues are shifted significantly from their corresponding resonance positions in the spectra of model polypeptides or apoferredoxin. Thirteen 13C resonances are shifted into the 80- to 120-ppm (from CS2) region, and have been assigned to the cysteinyl alpha and beta carbon atoms. The remaining shifted resonances in the 120- to 190-ppm region are tentatively assigned to amino acid residues that may be close to [4Fe-4S] clusters of the oxidized and reduced ferredoxins. The similarity in the shift pattern of the corresponding 13C resonances of the cysteinyl alpha and beta carbon atoms in the three ferredoxins studied suggests that the three-dimensional amino acid environments of the corresponding [4Fe-4S] clusters in each protein are similar.

Amino Acids↗
PubMed1978

Studies on the mechanism of formyltetrahydrofolate synthetase. The Peptococcus aerogenes enzyme.

Two conflicting mechanisms have been proposed for formyltetrahydrofolate synthetase (EC 6.3.4.3). Detailed studies with a clostridial enzyme support a sequential mechanism, while a stepwise mechanism with formation of a dissociable intermediate has been proposed for the Peptococcus aerogenes synthetase. However, the data supporting the P. aerogenes mechanism were obtained using synthetase of questionable purity and the results supporting the mechanism could be attributed to contaminating activities. Consequently, uncertainty still exists with regard to the enzyme mechanism. To resolve this uncertainty, the P. aerogenes formyltetrahydrofolate synthetase has been purified to homogeneity and used in experiments to reinvestigate the reaction mechanism. The results of P1:ATP, ADP:ATP, and formate:10-formyltetrahydrofolate exchange experiments as well as a steady state kinetic analysis revealed no difference in the mechanisms of the P. aerogenes or clostridial synthetases. The results are inconsistent with a stepwise mechanism involving a dissociable intermediate and consistent only with a sequential mechanism.

Clostridium↗
PubMed1977

Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins.

We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.

Amino Acids↗
PubMed1977

Ferredoxin and formyltetrahydrofolate synthetase: comparative studies with Clostridium acidiurici, Clostridium cylindrosporum, and newly isolated anaerobic uric acid-fermenting strains.

Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species, which contrasts sharply with their apparent high degree of phenotypic similarity.

Amino Acid Sequence↗
PubMed1976

Biosynthesis of ribothymidine in the transfer RNA of Streptococcus faecalis and Bacillus subtilis. A methylation of RNA involving 5,10-methylenetetrahydrofolate.

The methyl moiety of ribothymidine in the tRNA of Streptococcus faecalis, Bacillus subtilis, and probably Bacillus cereus is dervied from a 1-carbon folate derivative and not S-adenosylmethionine. Micrococcus luteus (M. lysodeikticus) tRNA appears to be almost devoid of ribothymidine. S. faecalis tRNA lacking ribothymidine can be methylated in vitro with formation of ribothymidine. 5,10-Methylenetetrahydrofolate has been identified as the 1-carbon donor involved in ribothymidine formation in S. faecalis and implicated in the analogous reaction in B. subtilis. The reaction requires FADH2 and/or another reducing agent present in cell extracts for the reduction of the methylene moiety.

Bacillus subtilis↗
PubMed1976

Formyl-methyl-methylenetetrahydrofolate synthetase-(combined). An ovine protein with multiple catalytic activities.

Formyltetrahydrofolate synthetase, methenyltetrahydrofolate cyclohydrolase, and methylenetetrahydrofolate dehydrogenase in sheep liver co-purify 200-fold to yield a homogeneous preparation containing a single protein species observed on discontinuous polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The synthetase and dehydrogenase activities migrate with the single protein band observed on polyacrylamide gel electrophoresis in two discontinuous buffer systems near pH 8. The protein sediments with a single symmetrical boundary with sedimentation coefficient (S20,w) = 8.30 S and elutes from molecular exclusion chromatography columns at a position corresponding to a diffusion coefficient (D20,w) = 3.99 X 10(-7) cm2 sec-1. Dodecyl sulfate-polyacrylamide gel electrophoresis of the protein intramolecularly cross-linked with dimethylsuberimidate shows one protein species of Mr = 218,000 in addition to the species of Mr = 108,500 characteristic of the unmodified protein. NH2-terminal analysis of the protein using 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chloride in the presence of dodecyl sulfate results in the recovery of a single dansyl amino acid, alanine. The three activities are thus shown to reside in a protein composed of two apparently identical subunits, and the trivial name formyl-methenyl-methylenetetrahydrofolate synthetase(combined) is proposed for this enzyme to suggest the multiple catalytic activities associated with the single protein species.

Adenosine Triphosphate↗
PubMed1976

Specificity of bacterial ribosomes and messenger ribonucleic acids in protein synthesis reactions in vitro.

Ribosomes from two Gram-negative bacteria translated f2 RNA, T4 early mRNA, mRNA from three Gran-negative bacteria, and mRNA from six Gram-positive bacteria; ribosomes from three Gram-positive bacteria translated mRNA from the Gram-positive strains, but did not translate the other mRNAs. Ribosomes from the Gram-negative bacterium Escherichia coli translated synthetic poly(U,G) but ribosomes from the Gram-positive bacterium Clostridium pasteurianum translated poly(U,G) very poorly, mRNA from Gram-negative bacteria was translated only in the presence of a high salt ribosomal wash containing initiation factors. mRNA from Gram-positive bacteria and synthetic poly(U,G) were translated much more efficiently when wash components were present, but were also translated to a small, but significant, extent in the absence of wash components. The translation specificity of each type of ribosome was independent of the source of ribosomal wash components. When the radioactively labeled products of in vitro protein synthesis were analyzed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography, it was found that each different bacterial and phage RNA preparation directed the synthesis of a unique set of polypeptide products of discrete sizes. Three different types of ribosomes were used to translate each of several Gram-positive bacterial messenger preparations; the overall patterns of products obtained with a given mRNA are similar, but some differences in the products formed or the relative amounts of the various products synthesized can be detected.

Azotobacter↗
PubMed1976

Derivatives of Clostridium acidi-urici ferredoxin containing altered amino acid sequences. Semisynthetic synthesis, biological activity, and stability.

The semisynthetic syntheses and some properties of derivatives of Clostridium acidi-urici ferredoxin that contain amino acid deletions or replacements in the peptide chain are described. All 16 stable derivatives prepared, with the exception of [Trp2]ferredoxin, were fully active as electron carriers in the two enzymatic assay systems tested: the phosphoroclastic system and the ferrodoxin-dependent reduction of cytochrome c. E1Trp1]Ferredoxin had 70% of the activity of native ferredoxin in both assay systems. The stability in aerobic solution of [Ala1]ferredoxin, which had had its natural alanyl NH2-terminal residue removed and then replaced chemically, is the same as that of the native ferrodoxin (half-life of approximately 54 days). The relative stabilities of derivatives with a replacement or deletion of the NH2-terminal residue are as follows: [Ala1]- greater than or equal to [Phe1]-, [Lys1]-, [ Pro1]-, [Leu1]- greater than [Met1]- greater than [Gly1]- greater than [Glu1]- greater than des-Ala1-ferrodoxin. The data indicate that a large bulky residue, but not a negatively charged residue, is tolerated in position 1 of the peptide chain and the greatly decreased stability (half-life = 1 day) of des-Ala1-ferredoxin confirms the importance of the NH2-terminal residue for the stability of the protein. The relative stabilities of derivatives containing Ala1, but including a replacement for the normal Tyr2, are as follows: Native greater than [Trp2]- greater than or equal to [Phe2]- greater than [His2]- greater than [Leu2]- greater than [Pro2]ferredoxin. [Gly2]- and des-Ala1-Tyr2-apoferredoxin did not form stable derivatives upon reconstitution with iron and sulfide, nor did [3-NO2-Tyr2, 30]- and [Leu2,3-NO2-Tyr30]apoferredoxins. Other relatively stable and fully active derivatives prepared included: [3-NH2-Tyr30]-, [3-F-Phe2]-, and [2-F-Phe2]ferredoxin. The behavior of these various derivatives demonstrates the importance of the peptide chain for the stability of C. acidi-urici ferredoxin and shows that the activity of ferredoxin can be altered by a single amino acid substitution in the peptide chain.

Amino Acid Sequence↗
PubMed1976

Apparent oxidation-reduction potential of Clostridium acidi-urici ferredoxin. Effect of pH, ionic strength, and amino acid replacements.

The effects of pH and ionic strength on the midpoint reduction potential (Emp) of Clostridium acidi-urici ferredoxin were determined using hydrogen gas and hydrogenase. The Emp of native ferredoxin at 24-25 degrees in 0.1 M Tris-chloride buffer, pH 7.0, is--0.434 V. In the pH range examined, the Emp becomes approximately 13 mv more negative per each pH unit increase. A plot of the log of ionic strength versus the apparent Emp of ferredoxin in 0.1 M Tris-chloride buffer, pH 7.5, Was linear over the range of 1.0 to 0.01 ionic strength with Emp values of--0.414 and--0.475 V, respectively, at these extremes. This effect is the same with sodium chloride, sodium bromide, or ammonium sulfate. Potassium phosphate buffer caused a similar change, but the absolute values of Emp differed from those obtained in the presence of the other salts. This effect of pH and ionic strength on Emp may be general for clostridial-type (Fe4S4)2-ferredoxins, since the apparent Emp of Clostridium pasteurianum ferredoxin is affected in a similar manner by these two variables. The Emp of this ferredoxin in 0.1 M Tris-chloride buffer pH 7.0, is--0.405 V. Since the NH2-terminal amino acid residue, Ala1, and Tyr2 of C. acidi urici ferredoxin are near an (Fe4S4)2-cluster in the protein, the apparent Emp of derivatives that contained amino acid replacements in these two positions were determined. Under similar conditions, the Emp of most of the 13 derivatives examined, including those of [Leu2]- and[3-NH2-Tyr30]ferredoxin, is approximately the same as that of native ferredoxin. However, the Emp of [His2]ferredoxin is approximately 15 mv more positive, whereas that of [Trp2]ferredoxin is 22 mv more negative than that of native C. acidi-urici ferredoxin. Variations in sodium chloride concentration and pH also affected the apparent Emp of the derivatives. It is suggested that the changes observed in the Emp of C. acidi-urici ferredoxin are caused by protein conformational changes.

Amino Acids↗