Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Ontogenetic studies on the topographical heterogeneity of somatostatin-containing neurons in rat hypothalamus

The ontogeny of the somatostatin-containing neuron system was investigated by light-microscopic immunohistochemistry. During development, immunoreactive somatostatin-containing neurons arise from three discrete regions of the neuroepithelium of the third ventricle and show a chronological difference. The neurons are first evident within the third ventricle floor on day 12.5 of gestation; they move thereafter to the arcuate nucleus. The second generation occurs in the dorsal region of the arcuate nucleus during days 17.5-19.5; these neurons migrate sequentially into the arcuate-ventromedial nuclear region. The third generation is recognized in the neuroepithelial cell layer of the rostral hypothalamus on day 17.5 of gestation; these cells move to the periventricular area. This latter generation is most prominent during days 3-6 after birth, and some of the cells are seen sporadically even up to day 20. The first two generations give rise to the somatostatin neuron system in the arcuate-ventromedial nuclear region, while the latter gives rise to that in the rostral periventricular region in the adult rat hypothalamus.     

Binding of N-acetyl-chitotriose to human lysozyme.

The interaction of N-acetyl-chitotriose ((GlcNAc)3) with human lysozyme [EC 3.2.1.17] was studied at various pH values by measuring changes in the circular dichroic (CD) band at 294 or 255 nm and the data were compared with the results for hen and turkey lysozymes reported previously (Kuramitsu et al. (1974) J. Biochem.76, 671-683; Kuramitsu et al. (1975) J. Biochem. 77, 291-301). The pH dependence of the binding constant of (GlcNAc)3 to human lysozyme was different from those for hen and turkey lysozymes. The catalytic carboxyls of human lysozyme, Asp 52 and Glu 35, were not perturbed on binding of (GlcNAc)3. This is consistent with the previous findings that the macroscopic pK values of Asp 52 and Glu 35 of human lysozyme are 3.4 and 6.8 at 0.1 ionic strength and 25 degrees and were unchanged on complexing with (GlcNAc)3. An ionizable group with pK 4.5, which participates in the binding of (GlcNAc)3 to hen lysozyme and was assigned as Asp 101, did not participate in the binding of the saccharide to human lysozyme. Between pH 9 and 11, the binding constants of (GlcNAc)3 to hen lysozyme remained unchanged, whereas perturbation of an ionizable group with pK 10.5 to 10.0 was observed for human lysozyme. This group may be Tyr 62 in the active-site cleft. The binding constants of (GlcNAc)3 to human lysozyme molecules having different microscopic protonation forms, with respect to the catalytic carboxyls, were estimated using the binding constants obtained in the present experiments and the microscopic ionization constants of the catalytic carboxyls obtained previously. All four species of human lysozyme had similar binding constants to (GlcNAc)3. This result is different from those for hen and turkey lysozymes.     

Rapid polarographic mutarotase assay with -D-glucose oxidase

This rapid mutarotase assay consists of polarographic measurements of rates of oxygen consumptions due to the enzymic oxidation with β-d-glucose oxidase of β-d-glucose formed from α-d-glucose in the absence and presence of mutarotase. This method requires only 5 min for each sample, and is applicable to aqueous solutions or suspensions with any turbidity and also to mutarotase samples containing large quantities of d-glucose. The mutarotase contents in rat tissues were determined by this method.     

An apparatus for thin layer vertical polyacrylamide gel electrophoresis with discontinuous buffer and gel system

A vertical polyacrylamide gel slab electrophoresis apparatus with a discontinuous gel and buffer system and a running gel of 1 mm in thickness was devised. Using this apparatus, which employs stacking and sieving effects, sharp bands comparable to those of disc electrophoresis were obtained.Furthermore, a new application using detection with ultraviolet method was introduced for isozyme study.     

Two single-base-pair substitutions causing desensitization to tryptophan feedback inhibition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum.

A 5-fluorotryptophan-resistant mutant, termed 1041, was isolated from Brevibacterium lactofermentum AJ12036. The anthranilate synthase of 1041 was insensitive to feedback inhibition by tryptophan, and the specific activities of the anthranilate synthase and anthranilate phosphoribosyltransferase of 1041 were 29- and 23-fold higher than those in parental strain AJ12036, respectively. A single-base change (adenine to cytosine) that resulted in a Ser-to-Arg substitution was found in the trpE structural gene of 1041. This substitution was identified as the cause of the desensitization to feedback inhibition by tryptophan of anthranilate synthase in 1041. Another substitution (guanine to adenine) was found at a position in which a mutation would destabilize the rho-independent terminator structure within the putative attenuator. The enhanced synthesis of tryptophan enzymes in 1041 could be caused by this substitution in the attenuator.     

Covalent binding of a mercaptan S-sulfate to hepatic cytosolic proteins and its inhibition by glutathione

4-Nitrobenzyl mercaptan (NBM) S-sulfate, a new type of the sulfate conjugate enzymatically formed from NBM in the presence of 3'-phosphoadenosine 5'-phosphosulfate in rat liver cytosol, bound covalently to rat liver cytosolic proteins at pH 7.4. The protein binding of NBM S-sulfate was strongly retarded by GSH. GSH not only played a role as a scavenger for NBM S-sulfate with formation of NBM and GSSG via S-(4-nitrobenzyl)thioglutathione, but also cleaved the covalent bonds, possibly disulfides formed from NBM S-sulfate and sulfhydryl groups of the cytosolic proteins. Thus, evidence was provided that NBM S-sulfate be a new type of the reactive metabolite.