Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by the phospholipase A2 from Agkistrodon halys blomhoffii venom

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the phospholipase A2 from the venom of Agkistrodon halys blomhoffii, was studied at 25 degrees C and the ionic strength of 0.1 in the presence of 3-33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the alpha-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799-804, and Haruki et al. (1986) J. Biochem. 99, 99-109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125-1130).(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes of papain induced on interaction with thiol proteinase inhibitors from newborn rat epidermis

The conformational changes of the papain molecular on interaction with two thiol proteinase inhibitors (TPI(1) and TPI(2] from newborn rat epidermis were studied by measuring circular dichroism (CD), the difference absorption spectrum, and the fluorescence spectrum due to tryptophan residues in papain. The far-ultraviolet CD band of papain between 210 and 230 nm was distinctly reduced on interaction with both inhibitors. Also, the near-ultraviolet CD spectrum of TPI(1)-bound papain changed between 285 and 320 nm as well as that of the TPI(2)-bound enzyme. The difference absorption spectrum for TPI(1)-bound papain exhibited two distinct peaks at 276.5 and 282 nm, indicating perturbation of aromatic amino acid residues. The fluorescence intensity of papain was significantly decreased on interaction with both inhibitors, which showed pH-dependency on an ionizable group, with pK values of 8.5 and 7.9 for TPI(1) and TPI(2), respectively. The complex formation of papain with both inhibitors caused a reduction of the susceptibility of a tryptophan residue, probably tryptophan-177, to chemical modification with N-bromosuccinimide. These results suggest that the active site involving histidine-159 in the papain molecule was much influenced by the alteration of the microenvironment of tryptophan-177 as a part of the interaction site for these two thiol proteinase inhibitors.     

An investigation for the occurrence of C4 photosynthesis in the Cyperaceae from Australia

Two hundred and twenty species of 38 genera in the Cyperaceae from Australia were examined for the possible occurrence of the C₄ photosynthesis and the anatomical features of leaves and culms. The Kranz type of anatomy and the carbon isotope ratios typical of C₄ plants were found in 84 species in the following six genera of four tribes belonging to subfamily Cyperoideae:Bulbostylis, Crosslandia, andFimbristylis (Fimbristylideae);Lipocarpha (Lipocarpheae);Cyperus (Cypereae);Rhynchospora (Rhynchosporeae). The anatomical observation revealed that the C₄ species possessed any one of the three Kranz anatomical types found by previous investigators. It was suggested that in the Cyperaceae the C₄ syndrome evolved independently within several taxa of the subfamily. The relative distribution of C₃ and C₄ species of the Cyperaceae in Australia was investigated by use of floristic data. It was recognized that the C₄ species dominated in the northern part of the continent which was characterized by tropical and subtropical savannas and hot dry areas with summer rainfall, and the C₃ species in the southern part, which contained temperate areas and mediterranean climatic areas with winter rainfall.     

Peripheral resistance and red cell LiNa countertransport in borderline hypertensives

We have studied ouabain-resistant, external sodium-stimulated, lithium efflux (LiNa countertransport) in red blood cells from 21 borderline hypertensives with at least one hypertensive first degree relative (BH-F), 19 borderline hypertensives without family history of essential hypertension (BH-NF), and 35 age-matched normotensive subjects. The data indicate the finding of an increased LiNa countertransport in all BH (F+NF), but with a significant overlap between BH values and control ones: LiNa countertransport is significantly higher only in BH-F but it is normal in BH-NF. Moreover, there is a significant correlation of LiNa countertransport to total peripheral resistance but not to mean blood pressure in all hypertensive patients. It is suggested that in BH the increase of erythrocyte Na flux is mediated by the NaNa exchange diffusion, and its abnormality may be associated to the hereditary trait of essential hypertension rather than the high blood pressure per se, probably resulting in the development of hypertension, through the increased vascular smooth muscle tone.     

Human laminin a chain (LAMA) gene: Chromosomal mapping to locus 18p11.3

Laminin, an integral component of basement membranes, consists of three subunit polypeptides, A, B1, and B2 chains. We have recently isolated cDNAs corresponding to human laminin A chain. These cDNAs were utilized for chromosomal in situ hybridizations to establish the genomic location of the laminin A chain gene. Metaphase chromosomes of PHA-stimulated human peripheral blood leukocytes were examined by in situ hybridization with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human laminin A chain is at locus 18p11.3. Since human laminin B1 and B2 chain genes have been previously mapped to chromosomes 7 and 1, respectively, the results indicate that genes encoding human laminin chains reside in separate chromosomes.     

Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.     

The fission yeast cut1+ gene regulates spindle pole body duplication and has homology to the budding yeast ESP1 gene

Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1+ and cut2+ genes are cloned by transformation. High gene dosage of cut1+ also complements cut2 and cut10 mutants. The cut2+ gene, however, complements only cut2. The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S. cerevisiae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.     

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8 A resolution.

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.