Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Kinetic Properties of Bovine Pineal Tryptophan-5-Monooxygenase Activated by an Endogenous Activating Substance

Abstract: We previously reported that an endogenous activating substance different from bovine serum albumin, phospholipids and heparin, exists in the extract from bovine pineal glands and that this substance interacts with tryptophan-5-monooxygenase under reducing conditions with sulfhydryl reagents, to stimulate monooxygenase activity. The present paper reports that the activating substance is of peptide nature; that it is sensitive to trypsin-digestion; and that it does not change the apparent K _m's for substrates, L-tryptophan and oxygen, and coenzyme, reduced biopterin or DMPH₄: but that it increases the V _max 1.5- to 2.3-fold. These results suggest that an activating protein, present in some particles of the cell structure, activates tryptophan-5-monooxygenase under the regulation of a sulfhydryl compound. The apparent K _m's for reduced biopterin and DMPH₄ were 77.2μM and 294 μM, respectively. The apparent K _m's for L-tryptophan and oxygen with reduced biopterin were 15.0 μM and 4.7%, respectively: with DMPH₄, they were 11.0 μM and 8.5%, respectively. Significant inhibition of both L-tryptophan and oxygen was observed with reduced biopterin, but not with DMPH₄ (at the tested concentrations of up to 0.5 MM and 20%, respectively).     

Kinetics of chemical modification of arginine and lysine residues in calf thymus histone H1

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.     

Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes

In a number of animal species soluble NADH-cytochrome b₅ reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b₅ reductase of liver microsomes by using an antibody to purified NADH-cytochrome b₅ reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b₅ and NADH-cytochrome b₅ reductase are also present in the ghost.     

Expression of cell adhesion molecule,Gicerin/CD146 during the formation of heart and in the cardiac hypertrophy

Molecular and Cellular Biochemistry - Gicerin/CD146 is a cell adhesion molecule which belongs to the immunoglobulin (Ig) superfamily. We have reported the existence of gicerin/CD146 in the nervous...     

Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.