Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.     

Metabotropic Glutamate Receptor Activation Produces Extrapyramidal Motor System Activation That Is Mediated by Striatal Dopamine

Little is known about the in vivo function of the GTP-binding protein-coupled "metabotropic" excitatory amino acid (EAA) receptor. In vitro studies on agonist-induced brain phosphoinositide hydrolysis have shown that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid is a highly selective and efficacious metabotropic EAA agonist. We have recently reported that in vivo unilateral intrastriatal injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid induces transient extrapyramidal motor activation that manifests itself as contralateral turning. In this study, we fully characterized the onset of turning behavior following intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid injection and the possible involvement of striatal dopamine neurons in the mediation of this effect. Rats were anesthetized with the short-acting agent halothane to allow for rapid surgical recovery and thus early behavioral measurements. Intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 mumol/2 microliters) produced an incremental increase in contralateral turning starting at 1 h and plateauing 3-6 h after injection (peak effect, 39.1 +/- 6.7 rotations per 5 min). Dopamine depletion with alpha-methyl-DL-p-tyrosine (250 mg/kg i.p., 80% depletion) resulted in greater than 85% inhibition of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced contralateral turning. The dopamine antagonist haloperidol (0.3 mg/kg i.p.) produced 48% inhibition of the (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid response. In time course studies, turning behavior correlated with increases in levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. These results suggest a functional interaction between the metabotropic EAA receptor and the dopaminergic system in the striatum.     

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane

Summary The mechanism of steroid uptake by the cell remains controversial. [³H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (K _d = 33 ± 4 nm, B _max = 32 ± 2 pmol/mg). [³H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 ± 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.This work was supported by grants PB85-0461 from the Comisión Asesora de Investigatión Científica y Técnica and PGV-8612 from the Departamento de Educatión, Universidades e Investigation del Gobierno Vasco. We thank Roussel-Uclaf (France) for the nonradioactive RU-steroids kindly provided.     

The shift from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by spermatozoa

There is a rapid shift in the steroidogenic pathway from androgen to progestogen production in spawning male common carp, Cyprinus carpio. Experiments were conducted to determine the mechanism regulating this shift using in vitro cultures of testicular fragments and isolated sperm of spermiating male carp. The levels of 11-ketotestosterone (11-KT) continually increased for 48 h with or without gonadotropin (GtH) stimulation, suggesting that 11-KT is the principal androgen produced by carp testes. Ovine prolactin (oPRL) enhanced GtH-stimulated 11-KT production, but by itself had no effect. Gonadotropin, carp pituitary extract, and pregnenolone all enhanced the production of 11-KT, testosterone (T), and 17 alpha-hydroxyprogesterone (17-P) in a dose-dependent manner. No 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was detected in response to any of these agents; 17 alpha,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P) was not measured. Both 17,20 beta-P and 17,20 alpha-P inhibited 11-KT production in a dose-dependent manner in the presence of either GtH, 17-P, or T. Isolated sperm and testicular fragment preparations both produced 17,20 beta-P and approximately tenfold more 17,20 alpha-P when incubated with 17-P. Only testicular fragments, however, produced 11-KT. We conclude that androgen synthesis occurs only within somatic cells of common carp testes. GtH, and perhaps PRL, stimulates the production of steroid precursors that, under normal physiological conditions, are metabolized to androgens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemical structure recognized by the L-fucose-specific hemagglutinin produced by Streptomyces sp

A hemagglutinin has been purified 4000-fold from the culture filtrate of a strain of Streptomyces by affinity chromatography. The purified preparation was judged to be homogeneous by gel electrophoresis and its molecular weight was estimated to be about 70 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It may exhibit its full hemagglutinating activity in the monomer form. This hemagglutinin strongly agglutinated human blood group O erythrocytes and was inhibited by L-fucose. It was, however, distinct from the known L-fucose-specific hemagglutinins; first, the hemagglutinating activity of the purified preparation was more than 100-times stronger than that of others; second, D-mannose was a potent inhibitor of this hemagglutinin besides L-fucose but not or scarcely inhibitory to others; and third, p-nitrophenyl-beta-L-fucoside was more inhibitory to this hemagglutinin than p-nitrophenyl-alpha-L-fucoside as opposed to the case of others.     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence     

Studies on Oxidative Fermentation. Part XII

Two substances besides rubiginol, of positive ferric chloride reaction were isolated from a shaking culture of Gluconoacetobacter liquefaciens, using glucose as the substrate. One of them was found to be a new γ-pyrone derivative and the composition was concluded to be 3,5-dihydroxy-l,4-pyrone-2-carboxylic acid. The other substance was found to be comenic acid. Assumptions were made with regard to the mechanism of the formation of these γ-pyrone derivatives from glucose.