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Nerve growth factor (NGF) is a neurotrophic factor that plays an important role in neuronal cell development and survival. Carnosic acid (CA), a hydrophobic constituent of the herb rosemary, induces NGF production in human T98G glioblastoma cells, but the mechanism through which it works remains unknown. In the present study, we found a redox-sensitive transcription factor, Nrf2, which coordinates the expression of cytoprotective phase 2 genes, also participates in CA-inducible NGF expression. In T98G cells, CA caused NGF gene induction in a dose- and time-dependent manner without altering NGF mRNA stability. Simultaneously, CA increased Nrf2 nuclear accumulation and activated expression of prototypical Nrf2 target genes such as haem oxygenase 1 (HO-1) and thioredoxin reductase 1 (TXNRD1). Knockdown of endogenous Nrf2 by Nrf2-specific siRNA significantly reduced constitutive and CA-inducible NGF gene expression. In addition, NGF gene expression was enhanced by knockdown of Keap1, an Nrf2 inhibitor, in the absence of CA. Furthermore, CA induced NGF expression in normal human astrocytes in an Nrf2-dependent manner. These results highlight a role of Nrf2 in NGF gene expression in astroglial cells.

Neurotrophins such as NGF promote neuronal survival and differentiation via the cell surface TrkA neurotrophin receptor. Compounds with neurotrophic actions that are low in molecular weight and can permeate the blood-brain barrier are promising therapeutic agents against neurodegenerative diseases such as Alzheimer's disease. Carnosic acid (CA), an electrophilic compound in rosemary, activates antioxidant responsive element (ARE)-mediated transcription via activation of Nrf2. In the present study, we discovered that CA strongly promotes neurite outgrowth of PC12h cells. NGF as well as CA activated Nrf2, whereas CA and NGF-mediated neuronal differentiation was suppressed by Nrf2 knockdown. On the other hand, CA activated TrkA-downstream kinase Erk1/2 independently of Nrf2. CA-induced p62/ZIP expression in an Nrf2-dependent manner, while the CA-induced neural differentiation was suppressed by p62/ZIP knockdown. Furthermore, CA-induced ARE activation was attenuated both by p62/ZIP knockdown and a Trk signal inhibitor. These results suggest that the CA induction of p62/ZIP by Nrf2 enhances TrkA signaling which subsequently potentiates Nrf2 pathway. This is the first demonstration that activation of the Nrf2-p62/ZIP pathway by a low-molecular natural electrophilic compound plays important roles in TrkA-mediated neural differentiation and may represent the common molecular mechanism for neurotrophic activities of electrophilic compounds.

We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.

4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent. Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.

A synthetic gene encoding 0.19 alpha-amylase inhibitor (alpha-AI) from wheat kernel was obtained by enzymatic assembly of 18 oligodeoxynucleotides which were chemically synthesized. The synthetic gene was introduced into vector pET15b for expression in Escherichia coli BL21(DE3) under the control of T7 promoter. However, in SDS-PAGE and Western blotting analyses of the E. coli cell lysate, the expression product could not be detected. Expression analysis for various partially deleted gene fragments suggested that the putative hairpin-like structure of mRNA in the 5'-terminal coding region might interrupt efficient expression. When the hairpin structure was eliminated by using degenerate codons, the resulting gene could be overexpressed in E. coli. Although the gene product was accumulated in an insoluble fraction as inclusion bodies, its inhibitory activity could be recovered by solubilization with 8 M urea, followed by refolding through two successive steps of dialysis at alkaline pH. After purification, the recombinant 0.19 alpha-AI showed the same characteristics as the authentic inhibitor in terms of N-terminal amino acid sequence, peptide mapping on reverse-phase HPLC, far-UV circular dichroism (CD) spectrum and have inhibition of human salivary alpha-amylase. Thus, we have established an overexpression system in E. coli for active recombinant 0.19 alpha-AI.

Chromosome fragmentation, ATP1 disruption, and Southern blot analyses of total DNAs and prime clones of chromosome II showed that three identical ATP1s are present, directing from the telomere to the centromere on the 35-55 kb far from the left telomere sequence of chromosome II. That is, the coding and 5'-, 3'-non-coding regions of ATP1 are repeated 3 times at approximately 7 kb intervals. These three ATP1s are expressed, and one and two ATP1s-disrupted strains, respectively, showed ca.70 and 40% decreases in their ATPase activities and alpha subunit contents, compared to those of the wild type, DC-5 or W303-1A strain, but could grow on glycerol.

H2-proteinase, a non-hemorrhagic metalloproteinase from the venom of Trimeresurus flavoviridis, has been crystallized by vapor diffusion from solutions containing ammonium sulfate. The crystals belong to the tetragonal space group, P41212 or P43212, with unit cell dimensions of a = b = 77.8 A and c = 82.3 A. The asymmetric unit contains one protein molecule. Diffraction data for a native crystal were collected up to 2.0 A resolution.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by Group I phospholipases A2 (PLA2s) from Pseudechis australis, Naja naja atra, and bovine pancreas and by Group II enzymes from Vipera russelli russelli, Agkistrodon halys blomhoffii, and Trimeresurus flavoviridis, were studied by the pH-stat assay method at 25 degrees C, pH 7.5-8.2, and an ionic strength of 0.1 or 0.2 in the absence or presence of an amide-type substrate analog, 2-dodecanoyl-amino-1-hexanol-phosphoglycol. The binding of genuine substrate to the Group II enzymes and that of its analog to the Groups I and II enzymes were markedly facilitated by the binding of Ca2+ to the enzymes. On the other hand, the binding of genuine substrate to the Group I enzymes was found to be independent of the Ca2+ binding. The former result suggests that the structures of the Group II enzyme-genuine substrate complexes and both types of enzyme-analog complexes are generally stabilized by the Ca2+ binding, whereas the latter indicates that the structures of the Group I enzyme-genuine substrate complexes are already similar to those of their Ca2+ complexes and that, therefore, these enzyme-substrate interactions are independent of the Ca2+ binding.

Six aldo-keto reductases from rat adrenal gland have been highly purified to apparent homogeneity. These enzymes were identified as carbonyl reductases (CR-K1, CR-K2, CR-A, and CR-B), aldehyde reductase (AR-H), and aldose reductase (AR-L) in terms of substrate specificity, molecular weight (33,000-39,000), inhibitor susceptibility, cofactor requirement, and immunochemical properties. Both CR-K1 and CR-K2 were characterized as possessing high affinity towards 13,14-dihydro-15-ketoprostaglandin F2 alpha and were localized immunohistochemically in the zona glomerulosa and the zona reticularis of adrenal cortex, and in the ganglion cell of adrenal medulla. Immunoreactive proteins to anti-CR-K2 antibody were observed in male and female reproductive tissues of rats. Positive immunoreactive protein to anti-CR-A antibody was found in mouse, hamster, and rabbit adrenal gland, whereas that to anti-CR-K2 antibody was present only in rat adrenal gland. AR-H and AR-L mainly reduced aromatic and aliphatic aldehydes. All the aldo-keto reductases from rat adrenal gland were completely inhibited by p-chloromercuribenzoate. Barbiturate and 3,3'-tetramethylene glutarate potently inhibited AR-H, and quercitrin significantly decreased the activity of CR-K1, CR-K2, and AR-L. We propose that these aldo-keto reductases may play important roles in the rat adrenal functions.

A hemorrhagic protein (60 kDa), HR1B, present in the venom of Trimeresurus flavoviridis is a mosaic protein consisting of an NH2-terminal metalloproteinase-domain, a disintegrin (platelet aggregation inhibitor)-like domain, and a unique COOH-terminal Cys-rich domain. Since the gross structures of HR1B and protein precursors of disintegrins, trigramin, and rhodostomin, all of which contain the metalloproteinase domain, are similar, many disintegrins so far detected in snake venoms are assumed to be autoproteolytic fragments released from precursors. In ongoing related experiments, the newly purified hemorrhagic metalloproteinases, HR1A from T. flavoviridis venom and HT-1 from Crotalus ruber ruber venom, in addition to HR1B, were autoproteolyzed, in the absence of Ca2+, at 37 degrees C for 3-12 h. Under these conditions, HR1A, HR1B, and HT-1 each released a single major fragment of 32, 34, and 31 kDa, respectively. The entire amino acid sequences of the isolated fragments indicated the presence of disintegrin-like and Cys-rich domains in the COOH-terminal regions of HR1A, HR1B, and HT-1, respectively. It seems likely that so-called disintegrins probably originate from various metalloproteinases present in venom. On the bases of peptide sequences close to the autoproteolytic cleavage sites of these metalloproteinases and the sites of fibrinogen cleaved by these enzymes, we synthesized new intramolecularly quenched fluorogenic peptide substrates. Among the 10 peptides tested, 2-aminobenzoyl (Abz)-Ser-Pro-Met-Leu-2,4-dinitroanilinoethylamide (Dna) proved to be the best substrate for venom metalloproteinase, as deduced from kinetic analyses.

The complete amino acid sequence of an antihemorrhagic factor, HSF, in the serum of the Japanese Habu snake, Trimeresurus flavoviridis, has been determined. The protein is composed of 323 amino acid residues and contains three asparagine-linked oligosaccharide chains at positions 123, 185, and 263. The molecule contains two copies of the cystatin domain in the N-terminal portion up to position 240, and these domains show a remarkable sequence homology (about 50%) to those of plasma glycoproteins such as alpha 2-HS (human) and fetuin (bovine) and to a lesser extent to that of HRG (human). The amino acid sequence of the noncystatin region towards the C-terminus is unique, showing no significant homology with those of the corresponding regions of alpha 2-HS and fetuin. In spite of the presence of cystatin domains, HSF does not inhibit cysteine proteinases such as papain and cathepsin B but does inhibit several metalloproteases in Habu venom. The results suggest that HSF is the first protein found to be functionally related to metalloproteinase inhibitors among the structurally homologous proteins with a double-headed cystatin domain, and is a member of a novel family (family 4) with divergent functions of the cystatin superfamily proteinase inhibitors. Although HSF possesses similar physicochemical properties to those of oprin, a snake venom metalloproteinase inhibitor with antihemorrhagic activity isolated from opossum serum [Catanese & Kress (1992) Biochemistry 31, 410-418], its primary structure is strikingly different from that of oprin.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by a cobra (Naja naja atra) (Group I) and a Habu (Trimeresurus flavoviridis) (Group II) PLA2s, were studied and compared with the results reported for other Group I and II enzymes. The substrate bindings to Group I enzymes were independent of the Ca2+ binding, whereas the substrate bindings to Group II enzymes were facilitated more than 10 times by the Ca2+ binding to the enzymes. The result for Group II enzymes, but not Group I enzymes, seemed compatible with the hypothesis for interpreting the catalytic mechanism that an intermediate complex should be stabilized by the coordination of the bound Ca2+ with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. The pH dependence of the kinetic parameters for the hydrolysis of the mixed micellar diC16PC, catalyzed by the cobra (N. naja atra) (Group I) and Habu (T. flavoviridis) (Group II) PLA2s, was also studied. The pK values of the catalytic group, His 48, and Tyr 52 for N. naja atra PLA2, shifted from 7.25 to 7.70 and from 10.30 to 10.85, respectively, and the corresponding values for T. flavoviridis PLA2 shifted from 5.80 to 6.95 and from 10.10 to 10.76, respectively, on binding of the micellar substrates to the enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method is presented for purifying antihemorrhagic factor in the serum of Japanese Habu snake (Trimeresurus flavoviridis). The method consists of specific binding of the factor to a hemorrhagic principle-conjugated Sepharose, gel filtration and reverse-phase HPLC. The improved method is simple and rapid, providing the factor with a great increase in specific activity and in a high yield.

The amino acid sequence of a cytotoxic factor, CTF-I, isolated from the venom of the Japanese habu snake (Trimeresurus flavoviridis) has been determined through automatic phenylisothiocyanate degradation of the PE-protein and derived proteolytic peptides. CTF-I consists of 72 amino acids and contains an Arg-Gly-Asp sequence present in trigramin-like peptides isolated from other snake venoms. The primary structure of another cytotoxic factor, CTF-II, consisting of 75 amino acids, was deduced to comprise that of CTF-1 with an additional Glu-Leu-Leu-sequence at its N-terminal.

A glutathione S-transferase isozyme which is absent in normal rat liver has been isolated from the hereditary hyperbilirubinuria rat liver cytosol. The enzyme was purified to apparent homogeneity by GSH-affinity chromatography and HPLC on CM-Sepharose CL-6B. It is a heterodimer of two non-identical subunits, i.e., subunit 2 and a previously uncharacterized subunit referred to here as subunit Yx. Immunoblot analysis indicated that GST 2-Yx belongs to the alpha class. GST 2-Yx is characterized by its 4-fold higher activity towards 4-hydroxy-non-2-enal, compared to that of GST 2-2.

The tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13,14-dihydro-15-keto-prostaglandin F2a, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a.

Caldesmon from chicken gizzard muscle has been examined for ability to interact with S100 protein using sedimentation, low-shear viscosity, and affinity chromatography. Ca2+/S100 protein, like Ca2+/calmodulin, inhibited the binding of caldesmon to F-actin in a concentration-dependent manner and the inhibition was not observed in the absence of Ca2+. Caldesmon was bound to S100 protein-Sepharose in the presence of Ca2+ and released with EGTA, indicating that there is a direct interaction between caldesmon and S100 protein. The binding of S100 protein to caldesmon also relieved actomyosin Mg2(+)-ATPase inhibition by caldesmon. The molar ratio of S100 protein to caldesmon required for half-maximal restoration was about 0.3, a value less than that in the case of calmodulin. S100 protein, however, was less effective in terms of the maximal extent of the restoration. With respect to Ca2(+)-sensitivity, the restoration profiles were monophasic with a midpoint at 3 x 10(-5) M for S100 protein and 8 x 10(-6) M for calmodulin. The restoration by S100 protein was almost wholly inhibited by TFP, but not by W-7. Taken together, our results suggest that a Ca2(+)-binding protein other than calmodulin may regulate caldesmon-dependent cellular functions.

The activities of hepatic cytosolic glutathione S-transferases (GSTs) towards 1,2-dichloro-4-nitrobenzene in male rats were higher than those in females, however, the enzyme activities towards 1-chloro-2,4-dinitrobenzene were not significantly different between the two sexes. SDS-PAGE analysis of GSTs purified from male and female rat hepatic cytosols by affinity column chromatography showed that there was a significant difference in the subunit composition between the two sexes. With regard to the several isozymes of GSTs in male and female rats, isozymes with basic and neutral/acidic isoelectric points were separated into seven molecular species by chromatofocusing. These sex differences in the quantitative proportions of GST isozymes were also confirmed by immunotitration using anti-GST-BL and -AC antibodies. On the other hand, glutathione peroxidase (GSH-Px) activities in rat hepatic cytosol towards hydrogen peroxide and cumene hydroperoxide were markedly higher in females than in males. Of the two types of GSH-Px, selenoenzyme (Se-GSH-Px) and the Se-independent enzyme (non-Se-GSH-Px), the former was found to be mainly responsible for the sex difference in the enzyme activities. Moreover, the GSH-Px activity of GSTs, non-Se-GSH-Px, was also higher in females than that in males. Since GST isozymes of the BL type are known to possess GSH-Px activity towards cumene hydroperoxide, the increased activities of non-Se-GSH-Px in the female hepatic cytosol seemed to be mainly due to the increased transferase activities of the isozymes, GST-L2 and -BL.

In order to identify the b-type cytochrome involved in the nitrate reduction in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, the b-type cytochromes in the spheroplast membranes were characterized. Difference spectra at 77K of spheroplast membranes indicated the presence of two b-type cytochromes with a bands at 556.5 and 562 nm. Three components considered to be of the b-type cytochrome were resolved by anaerobic potentiometric titration at 560-572 nm. Their midpoint potentials at pH 7, Em,7, were - 135 mV, +40 mV and +175 nm and their approximate reduced minus oxidized maxima were determined to be at 565 nm (562 nm at 77K), 560 nm (556.5 nm) and 560 nm (556.5 nm), respectively. These values are almost the same as those reported for R. sphaeroides. The Em,7 value of the cytochrome c involved in the nitrate reductase of this denitrifier was determined to be 250 mV. A b-type cytochrome reduced with NADH and FMN was oxidized by nitrate in chromatophore membranes. The possibility that cytochrome b (Em,7 = 175 mV) is involved in the nitrate reduction is discussed.

Age and sex differences in the hepatic glutathione level and related enzyme activities in rats of both sexes were investigated. At 7 weeks of age there was no significant difference in GSH levels between males and females, although a higher level of intracellular GSH was observed in male rats at 12 weeks of age. Hepatic gamma-glutamyl transpeptidase activities were significantly higher in females than in males at only 7 weeks of age. Glutathione peroxidase showed higher activities in females than in males. Conversely, glutathione S-transferase, glutathione reductase and glutathione synthesis rates were markedly higher in males than in females.

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.

Phospholipases A2 are classified into two groups, I and II, according to differences in the polypeptide-chain length and the intramolecular-disulfide bondings. The effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed and micellar phosphatidylcholines, catalyzed by a cobra (Naja naja atra) enzyme (Group I) and by mamushi (Agkistrodon halys blomhoffii) and habu (Trimeresurus flavoviridis) enzymes (Group II), were studied by the pH-statassay method at 25 degrees C, pH 8.0-8.2, and ionic strength 0.1-0.2. The results were compared with those reported for the other Group I and II enzymes. The Ca2+ binding was clearly shown to be essential for the catalysis of all the phospholipases A2. However, the substrate binding to Group I enzymes was found to be independent of the Ca2+ binding. On the other hand, the substrate binding to Group II enzymes was facilitated more than 10 times by the binding of Ca2+ to the enzymes. This was interpreted in terms of conformation changes of the peptide loop of residues 26 to 44 accompanying the Ca2+ binding. The latter result, but not the former, seems compatible with the hypothesis for interpreting the catalytic mechanism of phospholipases A2 that an intermediate complex should be stabilized by the coordination of the bound Ca2+ ion with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. According to the similarity in the primary and tertiary structures of the active sites of both types of enzymes [Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634], it is supposed that similar intermediate complexes may occur even for Group I enzymes, at least in the transition state of the productive complexes.

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.

When cells of the denitrifying phototrophic bacterium Rhodobacter sphaeroides forma sp. denitrificans were grown anaerobically under illumination in the presence of nitrate, the content of photosynthetic reaction centers per cellular protein was less than that in cells grown photosynthetically without nitrate under the same light intensity. The contents of cytochromes c1 and c2, which work in both photosynthetic and denitrifying electron transport systems, were almost constant, being independent of the presence of nitrate during growth. Consequently, the ratio of cytochromes c1 and c2 to the reaction center was more than three in the photo-denitrifying cells, whereas it was close to one in the photosynthetic cells under light-limiting conditions. In spite of the excess of cytochromes c1 + c2 over the reaction center in the photo-denitrifying cells, all cytochromes c1 + c2 were oxidized by illumination within hundreds of milliseconds in the presence of antimycin. When glycerol was added to increase the viscosity in the periplasm, biphasic oxidation of cytochromes c1 + c2 was apparent in the photo-denitrifying cells with repetitive flashes. The fast phase oxidation, which took place instantaneously (less than 1 ms) after the first and second flashes, showed a similar pattern to the oxidation in the light-limiting photosynthetic cells. The rate of the slow phase oxidation was sensitive to viscosity and was thought to reflect a diffusion-controlled second-order reaction between cytochrome c2 and the reaction center. The biphasic oxidation of cytochromes c1 + c2 suggests that these cytochromes exist in the photo-denitrifying cells as two different pools in relation to the reaction center.

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.

Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

The phospholipase A2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p-bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25 degrees C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1,2-bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal alpha-NH2 group had been selectively converted into an alpha-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the pKa value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the alpha-NH2 modified enzymes were also studied at 25 degrees C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (pKa 5.40) and His 48 (pKa 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the alpha-NH2 group (pKa 9.40) in addition to that of Asp 49 (pKa 5.40) and His 48 (pKa 7.53).(ABSTRACT TRUNCATED AT 250 WORDS)

Dimethylsulfoxide (DMSO) reductase was purified to electrophoretic homogeneity from the periplasmic fraction of a photodenitrifier, Rhodopseudomonas sphaeroides f.s. denitrificans. The enzyme had a molecular weight of 82,000 and had no subunit. It contained 1 mol of molybdenum per mol of enzyme, but iron and acid-labile sulfur were not present. The UV-visible spectrum showed only one absorption maximum at 280 nm. Denaturation of the enzyme released a molybdopterin cofactor, the fluorescence spectra of which were almost the same as those of a form B derivative of molybdopterin found in formate dehydrogenase. The Km value for DMSO was 15 microM, which was much lower than that for trimethylamin-N-oxide (TMAO), whereas Vmax with TMAO was larger than that with DMSO.

Several esters of a series of omega-amidino alpha-amino acids were synthesized. Some of them were shown to be efficiently hydrolyzed by bovine trypsin [EC 3.4.21.4]. The kinetics of hydrolysis of the esters were compared with those of the omega-guanidino series. The relationships between the length of the side chain and the susceptibility to hydrolysis by trypsin were somewhat different in the two series. These slight differences are discussed in terms of the molecular structures and their relation to the structural requirements of the trypsin active site.

Two carbonyl reductases have been highly purified from rat ovary to apparent homogeneity. Though they have similarities in terms of molecular weight (33,000), substrate specificities, inhibitor sensitivities, amino acid composition, and immunological properties, they differed in pI values (6.0 and 5.9). Both enzymes reduced aromatic aldehydes, ketones, and quinones at higher rates, compared to prostaglandins and 3-ketosteroids, whereas they showed higher affinity for prostaglandins and 3-ketosteroids. The enzymes also catalyzed oxidation of the 9-hydroxy group of prostaglandin F2 alpha. Moreover, they showed the remarkable characteristic of catalyzing the reduction of not only the 9-keto group of prostaglandin E2 but also the 15-keto group of 13,14-dihydro-15-keto-prostaglandin F2 alpha. Both enzymes were inhibited by SH-reagents, quercitrin, indomethacin, furosemide, and disulfiram. The results of immunoinhibition, using antibody against the purified enzymes, indicated that the enzymes were solely responsible for the overall catalytic activities of prostaglandin E series reduction, as well as 13,14-dihydro-15-keto-prostaglandin F2 alpha reduction and prostaglandin F2 alpha oxidation in rat ovarian cytosol. Western-blot analysis revealed that immunoreactive proteins were present in adrenal gland and various reproductive tissues except uterus of rats.