Abnormal S-state turnovers in NH3-binding Mn centers of photosynthetic O2 evolving system

The inhibitory effects of NH3 on S-state turnovers were studied by curve fitting and deconvolution of thermoluminescence glow curves and low-temperature EPR spectroscopy. The following results were found: (i) High concentrations of NH3 upshifted the recombination temperatures of both S2QB- and S2QA- charge pairs, indicating formation of an abnormal S2 state having a lowered oxidation potential. (ii) The abnormal S2 was correlated to alterations in EPR multiline signal: high concentrations of NH3 induced the modified multiline signal having reduced hyperfine line spacing, accompanied by disappearance of the g = 4.1 signal, while low concentrations of NH3 reduced the line width of the g = 4.1 signal with a slight shift in its g value to 4.2 concomitant with suppression in amplitude of the normal multiline signal, both suggesting coordination of NH3 to the Mn center. (iii) More than half of the NH3-binding abnormal S2 centers underwent S-state turnover to yield S3QB- and S3QA- pairs having normal thermoluminever, the NH3-binding S3 was unable to undergo further S-state turnovers. (iv) The interruption of S-state turnover at S3 was assumed to be due to the inability of electron abstraction from the S3 state. Based on these, the mechanism of NH3 inhibition was discussed.     

L-leucine and its analogue: specific inhibitors for S-benzyl-L-cysteine-p-nitroanilide-hydrolyzing enzyme in Escherichia coli B

An enzyme that catalyzes hydrolysis of S-benzyl-L-cysteine-p-nitroanilide was purified from E. coli B. The enzyme was a monomer with a molecular weight of 82,000. In addition to L-cysteinylglycine, the enzyme hydrolyzed various glycine-containing dipeptides most efficiently at pH 7.0. The enzyme required no metal ions for activity and was specifically inhibited by L-leucine and its analogue with free carboxyl group at the physiological concentrations.     

Role of sulphated tyrosine residue in influencing the biologic activity of human cholecystokinin-33

To evaluate the role of the sulphated tyrosine residue in position 27 in human cholecystokinin-33, parallel bioassay of the sulphated form of human cholecystokinin-33 and the unsulphated form of human cholecystokinin-33 was performed on the pancreatic protein secretion. Both peptides increased the protein output in a dose-related manner. However, the sulphated form possessed a considerably higher activity than the sulphated form. The relative potency of the unsulphated human cholecystokinin-33 compared to that of the sulphated human cholecystokinin-33 (taken as 1.0) was 0.08. From the results, it was suggested that the sulphated tyrosine may play an important role in controlling the activity of the longer molecular forms as well as that of the smaller forms of cholecystokinin.     

Comparative structures of the apopolysialoglycoproteins from unfertilized and fertilized eggs of salmonid fishes

The complete amino acid sequence of the major polysialoglycoproteins (PSGPs) from two genera of salmonid fish eggs, Salvelinus and Oncorhynchus, has been determined. The occurrence of tandem repeats of a genus-specific dodeca- and tridecapeptide was found for the apoPSGP of Salvelinus leucomaenis pluvius (Slp) and Oncorhynchus masou ishikawai (Omi), respectively, their amino acid sequences being highly homologous with that of rainbow trout [Salmo gairdneri (Sg)] apoPSGP (*denotes the glycosylation site; mean value of N = approximately 25): H-PSGP(Slp): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-)N H-PSGP(Omi): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser-)N H-PSGP(Sg): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly-)N Within 5-7 min following fertilization H-PSGP is converted to the low-molecular-mass PSGP (L-PSGP) by a specific protease (PSGPase). We have purified L-PSGP from the fertilized eggs of S. leucomaenis pluvius and Oncorhynchus keta (chum salmon) and compared it with rainbow trout egg L-PSGP(Sg) by analysis of their amino acid sequence: L-PSGP(Slp): Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp L-PSGP(Ok): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser L-PSGP(Sg): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly The data support the conclusion that H-PSGP is degraded in vivo 5-7 min after fertilization to L-PSGP by proteolytic cleavage at the position two residues C-terminally to the Pro residue, i.e., -Pro-Ser-Xaa-Asp-(Xaa = either Gly, Ser, or Asp) by the action of PSGPase.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.     

Identification of 2-azelaoylphosphatidylcholine as one of the cytotoxic products generated during oxyhemoglobin-induced peroxidation of phosphatidylcholine

Cytotoxic product(s), which are responsible for inducing the release of acetylcholinesterase-enriched vesicles from human erythrocytes and cell lysis, are generated when 1-saturated-2-polyunsaturated glycerophosphocholine was incubated with oxyhemoglobin (Itabe, H., Kobayashi, T. and Inoue, K. (1988) Biochim. Biophys. Acta 961, 13-21). To identify the products, a model compound, 1-O-octadecyl-2-linoleoylglycerophosphocholine was incubated with oxyhemoglobin. The oxidation products were isolated by both straight-phase and reverse-phase HPLC. The products, which were responsible for inducing erythrocyte membrane damage, were analyzed by secondary ion mass spectrometry and 1H-NMR. One of the cytotoxic products isolated was identified as 1-O-octadecyl-2-azelaoylglycerophosphocholine. Methyl esterification of the product confirmed the proposed structure.     

Interaction of surfactants with bilayer of negatively charged lipid: effect on gel-to-liquid-crystalline phase transition of dilauroylphosphatidic acid vesicle membrane

The interaction of surfactants with the vesicle membrane of the negatively charged lipid, dilauroylphosphatidic acid, was investigated through their effect on the gel-to-liquid-crystalline phase transition of the lipid bilayer. Three types of surfactants (anionic, cationic and non-ionic) with different hydrocarbon chain length were examined. (i) Anionic sodium alkylsulfates affected the phase transition temperature, Tm, only weakly. (ii) Non-ionic alkanoyl-N-methylglucamides decreased Tm monotonously with increasing concentration. The depression of Tm induced by these surfactants was analyzed by applying the van't Hoff model for the freezing-point depression, and the partition coefficients of the surfactants between bulk water and lipid membrane were estimated. (iii) Cationic alkyltrimethylammonium bromides affected Tm in a complex manner depending on the hydrocarbon chain length of the surfactants. Octyl-/tetradecyl-trimethylammonium bromide depressed/elevated Tm monotonously with increasing concentration, whereas the change in Tm induced by decyl- and dodecyltrimethylammonium bromides was not monotonous but biphasic. This complex behavior of the phase transition temperature was well explained, based on the statistical mechanical theory presented by Suezaki et al. (Biochim. Biophys. Acta, 818 (1985) 31-37), which takes into account the interaction between surfactant molecules incorporated in the lipid membrane.     

Pathological study on nephrocalcinosis in Fischer 344/Du Crj rats

Histopathological examinations on nephlocalcinosis of the Fischer 344 (F344) rats were carried out. As the results of comparison on its appearance among F344, Wistar and SD strains of rats, F344 female rats showed the most severe nephrocalcinosis. Nephrocalcinosis developed between 4 weeks and 8 weeks and was likely to keep its appearance through 108 weeks of the survival period of the rats. Histologically, mineral deposit was always observed at cortico-medullary junction. It seemed to locate at the outer portion of the basement membrane of the tubular epithelium, adjacent to the capillary wall in the connective tissue. Four weeks after ovariectomy at 4 weeks of age, the rats showed a decrease in degree of nephrocalcinosis. In contrary, the rats treated with estorone following ovariectomy revealed an increase in degree of nephrocalcinosis. It was suggested that the oestrogen-type sex hormone appeared to give a role in nephlocalcinosis.     

Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Mechanism of renal peritubular extraction of plasma glutathione. The catalytic activity of contralumenal gamma-glutamyltransferase is prerequisite to the apparent peritubular extraction of plasma glutathione

To clarify the peritubular mechanism for renal handling of plasma glutathione (GSH), variation of GSH levels in plasma, urine, kidney and liver was examined after intravenous administration of GSH to three groups of animals; control, acivicin-treated and rats treated with buthionine sulfoximine (BSO). Treatment of animals with BSO, a potent inhibitor of de novo GSH synthesis, markedly reduced hepatorenal GSH levels. Acivicin did not affect these levels. Upon intravenous injection of GSH (0.1 mmol/kg), renal GSH levels did not appreciably change in any of three animal groups. The rate of GSH disappearance from the circulation was rapid in control and BSO-treated rats, while it was markedly retarded in animals whose renal gamma-glutamyltransferase was extensively inactivated by acivicin. At 30 min after administration a significant amount of injected GSH was localized extracellularly (urine and plasma) in acivicin-treated animals. By contrast, most of the GSH rapidly disappeared from the extracellular space in control and BSO-treated animals. Together with the immunocytochemical evidence for the peritubular gamma-glutamyltransferase [Spater, H.W., Poruchynsky, M.S., Quintana, N., Inoue, M. & Novikoff, A.B. (1982) Proc. Natl Acad. Sci. USA 79, 3547-3550] the present results are fully consistent with the contention that the catalytic function of this enzyme is principally responsible for the peritubular mechanism for the renal handling of plasma GSH.