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J Sy

Publications and source records attributed to J Sy.

35 records · Page 2Linked to original sources
PubMed1976

Purification and properties of guanosine 5', 3'-polyphosphate synthetase from Bacillus brevis.

A ribosome-independent guanosine 5',3'-polyphosphate synthetase has been highly purified from Bacillus brevis (ATCC 8185). The enzyme has a molecular weight of 55,000, as measured by sucrose density gradient centrifugation. Like the ribosome-connected stringent factor of Escherichia coli, it catalyzes the synthesis of the guanosine 5', 3'-polyphosphates by a pyrophosphoryl transfer mechanism from adenosine triphosphate (ATP) to guanosine di- or triphosphates (GDP, GTP). It has an apparent Km of 0.14 mM for GDP and 0.77 mM for GTP, and is specific for the guanosine ribonucleotides as pyrophosphoryl acceptors. Several ATP analogues were tested for their ability to donate the pyrophosphoryl group. Mg2+ was required as a counter ion for the nucleotide substrate; however, an excess of Mg2+ was inhibitory. The property of the B. brevis enzyme is compared with the ribosome-linked enzyme of E. coli and an extracellular enzyme excreted by several types of Streptomyces reported upon recently.

Bacillus↗
PubMed1976

A ribosome-independent, soluble stringent factor-like enzyme isolated from a Bacillus brevis.

A ribosome-independent synthesis of guanosine 5',3'-polyphosphates has been found in the soluble fraction of Bacillus brevis (ATCC 8185) extracts. The partially purified enzyme catalyzes the formation of both guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate, does not require 20% methanol to stimulate the rate of reaction, and is not stimulated by complexing with ribosomes of either Escherichia coli or B. brevis. The B. brevis enzyme system is not inhibited by RNase A or thiostrepton, and is only slightly inhibited by tetracycline. The pyrophosphoryl donor specificity of the B. brevis enzyme is similar to that of the E. coli ribosome-stringent factor system.

Bacillus↗
PubMed1975

Nucleotide specificity of stringent factor and the synthesis of analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate.

The ribosome-dependent stringent factor reaction was found to be nonspecific with regard to the number of phosphate groups linked to the 5' position of guanosine nucleotides. Both GMP and guanosine 5'-tetraphosphate could accept a pyrophosphoryl group from ATP although at a much lower rate than GDP or GTP. Guanosine 5'-monophosphate 3'-diphosphate and guanosine 5'-tetraphosphate 3'-diphosphate were the products of these reactions. Furthermore, 3'-linked analogs of guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate were synthesized from the corresponding ATP analog, adenosine 5'-O-(3-thiotriphosphate). The stringent factor catalyzed reverse reaction was found to be specific for guanosine 5'-diphosphate 3'-diphosphate, and was essentially inactive to the isomeric form, guanosine 5'-diphosphate 2'-diphosphate.

Bacterial Proteins↗
PubMed1974

Reversibility of the pyrophosphoryl transfer from ATP to GTP by Escherichia coli stringent factor.

The stringent factor-catalyzed, ribosome-dependent synthesis of guanosine polyphosphates is found to be reversible. The reverse reaction specifically requires 5'-AMP as the pyrophosphoryl acceptor, and guanosine 5'-triphosphate-3'-diphosphate is preferentially utilized as the pyrophosphoryl donor. The primary products of the reaction are GTP and ATP. The reverse reaction is strongly inhibited by the antibiotics thiostrepton and tetracycline, and by ATP and beta-gamma-methylene-adenosine-triphosphate, but not by ADP, GTP, and GDP. The reverse reaction occurs under conditions for nonribosomal synthesis. The overall reaction for stringent factor-catalyzed guanosine polyphosphate formation may thus be formulated: (p)pp5'G + ppp5'A right harpoon over left harpoon (p)pp5'G3'pp + p5'A.

Adenosine Diphosphate↗
PubMed1973

Identification of the synthesis of guanosine tetraphosphate (MS I) as insertion of a pyrophosphoryl group into the 3'-position in guanosine 5'-diphosphate.

The phosphate transfer system of Haseltine et al., consisting of a ribosomal wash obtained from a stringent strain of Escherichia coli, washed ribosomes, GTP, and ATP, was used to prepare large quantities of guanosine tetra- and pentaphosphates, the magic spot compounds MS I and MS II of Cashel and Gallant. In our hands, the Haseltine et al. system yielded predominantly guanosine tetraphosphate, ppGpp. This system was used exclusively in the described experiments, with ATP labeled with (32)P in the beta- and gamma-positions as donor. The beta-label was found to produce a ppGp⃰p and the gamma-label a ppGpp⃰. Furthermore, [(3)H]GDP + [gamma-(32)P]ATP yielded ppGpp in a (3)H:(32)P ratio of 1:1. The results indicate a transfer of the terminal pyrophosphoryl group of ATP as a unit. The position of the transferred pyrophosphoryl was assayed for by preparation of pGp⃰ from ppGp⃰p with Zn(++)-activated inorganic pyrophosphatase from yeast. The pGp⃰ was then assayed with 3'-nucleotidase, which liberated practically all the labeled phosphate. This result indicate that the phosphate transfer from ATP to GDP yields guanosine 5'-diphosphate-3'-diphosphate.

Adenosine Triphosphate↗
PubMed1973

Nonribosomal synthesis of guanosine 5',3'-polyphosphates by the ribosomal wash of stringent Escherichia coli.

A factor in the ribosomal wash of stringent strains of E. coli was identified by Haseltine et al. as a complement to the ribosomal system for the synthesis of the magic spot compounds of Cashel and Gallant. This factor has been found, in the absence of ribosomes, to catalyze the enzymatic pyrophosphoryl transfer from ATP to GTP or GTP in the formation of magic spot I and magic spot II, the guanosine tetra- and pentaphosphates (ppGpp and pppGpp), respectively. The enzyme, which normally requires the presence of the ribosome-tRNA-mRNA complex for activity, catalyzes a very slow synthesis that is stimulated tenfold by 20% methanol. The temperature optimum of the methanol-stimulated system is 25-30 degrees and activity is drastically depressed at 37 degrees , presumably by inactivation. Catalysis is linear with enzyme concentration and with time for the first 3 hr; during this period 25% of the added GTP is converted. The nonribosomal system is distinguished from the ribosomal system by having a lower Mg(++) and a higher NH(4) (+) optimum. The two systems differ in their response to antibiotics: thiostrepton strongly inhibits the ribosomal system but has no effect on the nonribosomal system.

Ammonium Chloride↗