PubMed Health⌕ Search

Biomedical subjects

T Kurotsu

Publications and source records attributed to T Kurotsu.

25 records · Page 2Linked to original sources
PubMed1981

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis.

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.

Amino Acid Isomerases↗
PubMed1981

Sulfhydryl groups related to the catalytic activity of gramicidin S synthetase 1 of Bacillus brevis.

Gramicidin S synthetase 1 (GS 1) [EC 5.1.1.11] (phenylalanine racemase) of Bacillus brevis contained about six sulfhydryl groups as determined by titration of the enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). Two types of sulfhydryl groups could be detected in the reaction with DTNB. One sulfhydryl group reacted rapidly with DTNB whereas the other five reacted more slowly with it. Phenylalanine racemizing activity was abolished on the rapid sulfhydryl modification with DTNB. When GS 1 of the wild strain was preincubated with phenylalanine at 37 degrees C in the presence of ATP, MgCl(2), and dithiothreitol (DTT), the rapid sulfhydryl modification with DTNB was prevented. When GS 1 was incubated with L-[14C]phenylalanine in the presence of ATP, MgCl(2), and DTT, 1 mol of L-[14C]phenylalanine was incorporated per mol of enzyme protein as an acid-stable phenylalanine thioester-enzyme complex. On the other hand, for GS 1 of a gramicidin S non-producing and phenylalanine racemization-lacking mutant of B. brevis, the substate protection against the rapid sulfhydryl modification was not detected and L-[14C]phenylalanine was not incorporated into the enzyme protein as the thioester complex. These results strongly suggest that one sulfhydryl group of GS 1 which reacts rapidly with DTNB is essential for the racemizing activity.

Adenosine Triphosphate↗
PubMed1978

Studies on gramicidin S synthetase. Purification of the heavy enzyme obtained from some mutants of Bacillus brevis.

The heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20,W value of 12.2 at a protein concentration of 2.5 mg per ml. These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.

Amino Acid Isomerases↗
PubMed1978

Studies on gramicidin S synthetase. Purification and properties of the light enzyme obtained from some mutants of Bacillus brevis.

The phenylalanine-activating and/or-racemizing enzyme, i.e., the light enzyme, of gramicidin S synthetase was purified to a homogenous state by D-phenylalanine-Sepharose 4B chromatography from a wild and some gramicidin S-lacking mutant strains of Bacillus brevis. The light enzyme obtained from a mutant strain E-1 could activate phenylalanine but not racemize it, and had no phenylalanine-dependent ATP-[14C]AMP exchange activity, whereas the same enzyme obtained from other mutants and the wild strain had all three activities. Furthermore, the light enzyme of the mutant E-1 could form only acid-labile enzyme-bound phenylalanine, while the same fraction of the wild strain carried half of the enzyme-bound phenylalanine as acid-labile adenylate and half as a acid-stable thioester. These results suggest that the thiol site of the light enzyme of mutant E-1 might be damaged.

Amino Acid Isomerases↗