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.     

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.     

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.     

DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography.

A new method of affinity chromatography using blue dextran-Sepharose 4B resin was established to purify NADP+-dependent isocitrate dehydrogenase [EC 1.1.1.42] from Bacillus stearothermophilus in high yield. The purified preparation was found to be homogeneous on disc gel electrophoresis. The SH groups of the enzyme were modified with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to determine the number of SH groups per molecule and their contribution to the enzyme activity. One SH group was titrated with DTNB per subunit (the native enzyme consisted of two subunits) and after complete denaturation with 4 M guanidine-HCl the number of titratable SH groups remained unchanged. ORD and CD measurements showed that the alpha-helical conformation of the polypeptide backbone was unaffected by DTNB modification, though the near ultraviolet CD spectrum was evidently altered. The fluorescence derived from tryptophanyl residue(s) was quenched by the modification to 30% of the native level, which may indicate the presence of SH in the vicinity of tryptophanyl residue(s). A remarkable decrease of the enzyme activity was detected upon modification with DTNB, but there was some discrepancy between the rate of inactivation and that of modification of SH groups. The presence of substrate and Mg2+ gave partial protection against modification of the SH groups by DTNB. Complete protection of the native enzyme activity against heating at 65 degrees was observed in the presence of substrate and Mg2+, but the thermostability of the enzyme was markedly reduced by modification of the SH groups.     

Molecular adaptability of carp myosin: a study of some physico-chemical properties and their comparison with those of rabbit myosin.

During thermal inactivation, the addition of as low as M urea resulted in the reduction of delta G identical to barrier of the inactivation of carp myosin Ca2+-ATPase, whereas that of rabbit myosin remained unaffected. In the absence of urea, a four-hour incubation of carp myosin was accompanied by the release of light chains at 30 degrees C, a value 10 degrees C lower than that for rabbit myosin. Electron micrographs revealed that carp myosin forms artificial thick filaments, which were uniform in size and may differ in a few details from those of rabbit. Not only that helical content of carp myosin was about 4% less than those of rabbit myosin, but it showed more sensitivity to thermal and urea denaturation; and its reversibility upon subsequent cooling or removal of urea was rather poor. The loss in helicity of myosins by urea was a concentration- and temperature-dependent biphasic reaction, with the most obvious effect observed on carp myosin. That carp myosin has increased tendency of unfolding in urea solutions was confirmed by viscosity data and the exposure of thiols also. Even in the absence of urea more SH groups of carp myosin were incorporated by DTNB, and more epsilon-amino groups reacted with NQS. Carp myosin remained in solution till the modification of about 52 surface myosin remained in solution till the modification of about 52 surface amino groups, whereas no precipitation effect was noted in case of rabbit myosin. Neither amino-acid composition nor some parameters derived from it, such as average hydrophobicity polarity index and number of polar side chains, revealed any difference pertinent to the relative stability of the two myosins. On the contrary, the contractile efficiency of carp myosin in the near physiological range was high and thus inversely related with the thermostability. This relationship along with the above evidence has been regarded to demonstrate the adaptability of carp myosin through a loose molecular conformation, which has probably been achieved by the addition of weak interactions in the course of evolution.     

Comparison of the gamma-crystallins isolated from eye lenses of shark and carp. Unique secondary and tertiary structure of shark gamma-crystallin

gamma-Crystallin isolated from the shark of cartilaginous fishes was compared with the cognate gamma-crystallin from the carp of bony fishes. Distinct differences in amino acid compositions, primary, secondary and tertiary structures were found. The most salient features of shark gamma-crystallin lie in the fact that this crystallin possessed a significant alpha-helical structure in the peptide backbone as revealed by circular dichroism study, in contrast to those orthologous gamma-crystallins from other vertebrate species including bony fishes which all show a predominant beta-sheet secondary structure. The tertiary structure as reflected in the intrinsic microenvironments of various aromatic amino acids in the native crystallins also shows unambiguous differences between these two classes of gamma-crystallins. N-Terminal sequence analysis corroborates the structural differences between shark and carp gamma-crystallins. gamma-Crystallin from the more primitive shark seems to be more in line with the main evolutionary phylogeny leading to the modern mammalian gamma-crystallin.