The expression of each of the Escherichia coli ompF and ompC genes is activated by the common positive regulator, OmpR, in response to the medium osmolarity. The promoters for these genes consist of canonical -35 and -10 regions, and upstream OmpR-binding sites. In this study, we constructed a functional ompF-ompC chimeric promoter consisting of the OmpR-binding site of ompF, and the -35 and -10 regions of ompC, which was fused to the lacZ gene on a low-copy-number plasmid. It is known that the ompF and ompC genes are expressed in a different manner in cells with mutations in the ompR gene. In this respect, it was found that the ompF-ompC chimeric promoter behaves just like ompF promoter with respect to an ompR mutation (ompR472). It was revealed that, in this chimeric promoter, the OmpR-binding site must be located stereospecifically with respect to the -35 and -10 regions for OmpR-dependent transcription of the ompF-ompC chimeric promoter to occur. These results are discussed in relation to the structures and functions of the ompF and ompC promoters, the occurrence of a DNA curvature in the promoter regions being implied, which may be an important parameter for the transcription activation by the OmpR protein.

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With bovine myelin basic protein as a model common substrate, protein kinases C (PKC) purified from yeast (Saccharomyces cerevisiae) and mammalian tissue (rat brain) were shown to exhibit clearly different catalytic properties. The major sites of phosphorylation in bovine myelin basic protein by the yeast PKC were identified: Thr-19, Thr-34, and Thr-65. These sites are distinctly different from those for the mammalian PKC: Ser-8, Ser-46, Ser-55, Ser-110, Ser-132, Ser-151, and Ser-161, which were previously identified [Kishimoto, A., Nishiyama, K., Nakanishi, H., Uratsuji, Y., Nomura, H., Takeyama, Y., & Nishizuka, Y. (1985) J. Biol. Chem. 160, 12492-12499]. The results suggest that the yeast and mammalian enzymes may play distinct roles in cellular regulation. No evidence is available, however, that a yeast-type PKC exists in mammalian tissues. An oligopeptide containing the sequence around Thr-19 of bovine myelin basic protein, Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr (19)-Met-Asp-His-Ala, can be used as a substrate for selective assaying of the yeast PKC.

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The complete amino acid sequence and disulfide-bridge location of canine haptoglobin (Hp) were determined by analyzing various fragments produced chemically and/or enzymatically. Canine Hp consists of two light (L) and two heavy (H) chains with 83 and 245 amino acid residues, respectively. It has three potential oligosaccharide-binding sequences, Asn-X-Ser/Thr, one in the L chain and two in the H chain. All of them are glycosylated. Comparison of the amino acid sequences between canine Hp and human Hp shows 68 and 85% homology for L chains and H chains, respectively. About 20% of the canine L chain still retains a carboxyl-terminal arginine residue, which is completely removed during maturation in human L-chain. The half-cystine residue at position 15 in the L chain, which participates in the inter-L chain disulfide bridging in human Hp, has been replaced by a leucine residue in canine Hp. Therefore, an LH unit in canine Hp may be joined to another LH unit by a noncovalent (mainly hydrophobic) interaction to form the complete molecule. The disulfide bridges in canine Hp link Cys-34L to Cys-68L, Cys-72L to Cys-105H, Cys-148H to Cys-179H, and Cys-190H to Cys-220H, as in the case of human Hp.

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Diphosphate-modified substrates for prenyltransferase were synthesized and examined as substrates for the prenyltransferase reaction. They were dimethylallyl methylenediphosphonate, geranyl methylenediphosphonate, geranyl imidodiphosphate, geranyl phosphosulfate, farnesyl methylenediphosphonate, farnesyl imidodiphosphate, and farnesyl phosphosulfate. All of them except dimethylallyl methylenediphosphonate were accepted as substrates by solanesyl diphosphate synthase to give solanesyl diphosphate and the former four analogs were also accepted as substrates by farnesyl diphosphate synthase to give farnesyl diphosphate. The K_m values of both enzymes for the methylenediphosphonate and imidodiphosphate analogs were comparable to those of the corresponding diphosphate substrates, but the phosphosulfate analogs showed much greater K_m values than the diphosphate substrates. On the other hand, the V_max values for these artificial substrates were all smaller than those for the corresponding natural substrates. Kinetic experiments with the analogs showed that the ionization-condensation-elimination mechanism proposed for the farnesyl diphosphate synthase reaction holds also for the solanesyl diphosphate synthase reaction and that the diphosphoryl structure, capable of chelating with divalent cations, is important topologically and kinetically rather than thermodynamically.

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We have isolated a cDNA clone encoding ubiquitin carboxyl-terminal hydrolase PGP9.5 from a rat brain cDNA library and examined the tissue distribution. The primary structure of the cDNA consists of 856 nucleotides including the entire coding region for 223 amino acids, and the calculated molecular mass is 24, 782 Da. The rat PGP9.5 is strikingly homologous to the human PGP9.5, 75.2% of nucleic acids and 95.1% of amino acids being identical. The mRNA of PGP9.5 is most abundant in the rat brain and to a lesser degree in the testis. In other peripheral tissues we tested, the mRNA was undetectable. Western blotting using an anti-rat PGP9.5 antibody revealed the parallel distribution of mRNA and protein in various brain regions and testis. The availability of the rat PGP9.5 clone provides a new approach to examine the function of PGP9.5 and the role that it plays in the pathology of neurodegenerative diseases.

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A (Ca²⁺-Mg²⁺)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a K_m value for ATP of 0.17mM and a V_max of 71.4μmol/min/mg protein at 37°C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca²⁺ and Mg²⁺ on the ATPase were not additive, thereby indicating that both Ca²⁺ and Mg²⁺-ATPase activities are manifested by the same enzyme. The (Ca²⁺-Mg²⁺)-ATPase differed from H⁺-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca²⁺-Mg²⁺)-ATPase were determined.

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The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides n-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than n-glucose. The enzyme showed higher activity with β-D-glucose than with the α-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. β-D-Cellobiose was also phosphorolyzed faster than the α-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with α-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.

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The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of β-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U//α, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the α-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (>2mg/liter), easy purification and needlessness of disulfide refolding.

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The levels of aspartase, NADP- and NAD-requiring glutamate dehydrogenases (GDHs) in Pseudomonas fluorescens grown under various nutritional conditions were determined. NADP-GDH showed the highest value on glucose-ammonium sulfate medium and markedly lower values on amino-acid and casamino-acids media, while the reverse was found for the NAD-GDH, as in the case of other microorganisms with two GDHs. Aspartase did not show a marked variation between the media examined. Glucose nutritionally induced NADP-GDH but suppressed NAD-GDH; and it had no effect on aspartase, which was slightly induced by casamino acids. Transfer of the cells grown on glucose-ammonium sulfate medium to casamino-acids medium clearly increased the levels of NAD-GDH and aspartase, while addition of chloramphenicol to the media abolished the increases, suggesting that the increases were due to de novo synthesis of the enzyme proteins. These results indicate that the aspartase of this microorganism has a different function from those in others, including Escherichia coli.

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Expression of a fusion protein composed of dihydrofolate reductase and a derivative of growth hormone-releasing factor resulted in the formation of inclusion bodies in Escherichia coli at 37°C. Among various chemicals, such as detergents, protein denaturants, and acetic acid, tested for the ability to dissolve the inclusion bodies, acetic acid, Brij-35, deoxycholic acid sodium salts, guanidine-HCI, and urea showed a strong solubilizing effect without damaging the DHFR activity. Acetic acid was useful in terms of preparing GRF derivatives, since it could be easily removed by lyophilization, and this made it easy to perform the succeeding BrCN treatment for cutting out the GRF derivative from the fusion protein. The GRF derivative was purified by reversed phase HPLC from the BrCN digest of the acetic acid extract, and its growth hormone-releasing activity was demonstrated. However, for obtaining a highly purified fusion protein itself, solubilization of inclusion bodies by urea was preferred because urea was the only agent which did not cause serious precipitation of the regenerated fusion protein after 10-fold dilution of the extracted inclusion bodies with buffer. The fusion protein was highly purified by means of a methotrexate affinity chromatography.

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The ciliary epithelium is a bilayer of epithelial cells responsible for the formation and secretion of aqueous humor in the mammalian eye. We have isolated a cDNA clone from a λgt11 cDNA library of bovine ocular ciliary epithelial cells encoding the CD9 antigen, a member of a new family of transmembrane proteins. The bovine CD9 clone contains an open reading frame of 226 amino acids (M_r 24, 860). The deduced amino acid sequence from the bovine CD9 cDNA clone shows 83.5% identity with the human counterpart isolated from megakaryocytes, and a lower degree of identity with a group of related antigens (TAPA-1, C0-029, CD53, MRC OX-44, ME491, CD63, CD37, and Sm23) involved in growth regulation. Analysis of bovine ocular tissues reveals that the CD9 gene encodes a 1.4 kb mRNA which is detectable predominantly in cornea and at low levels in ciliary epithelium, retina, iris, and lens. Normal and transformed cell lines established from the ocular ciliary epithelium exhibited significant levels of CD9 transcripts. These results raise questions regarding possible roles of CD9 in the anterior segment of the eye.

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The structure of the Asn-linked carbohydrate chain of batroxobin, a thrombin-like enzyme from Bothrops atrox moojeni snake venom, has been determined. The sugar chain was isolated from batroxobin by hydrazinolysis followed by pyridylamination (PA). The PA-oligosaccharide chain was purified by HPLC on an anion exchange or reverse phase columns, and its structure was examined by sequential exoglycosidase digestion, 600MHz ¹H NMR spectroscopy and methylation analysis. The results indicate that the oligosaccharide chain has the following structure involving a novel linkage, NeuAcα2→3GalNAc.
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An alternative method for amplification of DNA with unknown sequences was developed. This involves the direct ligation of a primer oligodeoxyribonucleotide itself to restricted DNA fragments with unknown sequences to be amplified by PCR. The oligonucleotide need not be phosphorylated and need not be annealed with its complementary oligonucleotide in advance for ligation. The ligation reaction seems to be independent of the concentration of unknown DNA, proceeds in short time, and is efficient. The ligation efficiency was more than 30% at a low concentration, 10fg/μl, of DNA. This method was applied to a microdissection/microcloning of the short arm of human chromosome 2. Of 65 clones screened for the highly repetitive sequences with total human genomic DNA, eleven (17%) were positive. Their inserts ranged in size from 150 to 1, 200 bp (average, 460 bp). In Southern blot analysis, thirty consecutive clones all detected signals common to both total human genomic DNA and mouse-human hybrid cell DNA containing only chromosome 2 of human origin. Among them, 24 (80%) were unique sequences, and 6 (20%) were multi-copy (or intermediate-repeat) sequences. Thus, this method is simple and efficient, and provides an alternative way to amplify unknown DNA.

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In order to clarify the biological activities of (-)-oxetanocin G, and (-)-oxetanocin A and its carbocyclic analogue, (-)-carboxetanocin G, the inhibitory effects of triphosphate derivatives of these compounds (OXT-GTP, OXT-ATP, and C-OXT-GTP) on eukaryotic and viral DNA polymerases were examined. DNA polymerase α purified from calf thymus was weakly inhibited by OXT-GTP and OXT-ATP but strongly by C-OXT-GTP, the K₁ value being 0.22μM. On the other hand, rat DNA polymerase β was not affected by these analogues. DNA polymerase γ purified from bovine testes was very weakly inhibited by OXT-GTP and OXT-ATP, but not by C-OXT-GTP. DNA polymerase from herpes simplex virus type-II (HSV-II) was strongly inhibited by all three analogues, the K₁ values ranging from 0.5 to 1.0μM. Human immunodeficiency virus-encoded reverse transcriptase (HIV RT) was also strongly inhibited by these three analogues, the K₁ value of C-OXT-GTP being slightly smaller than that of OXT-GTP or OXT-ATP. Analysis of products synthesized on singly primed M13 single-stranded DNA by DNA polymerase α, HSV-II DNA polymerase or HIV RT in the presence of the analogues revealed that OXT-GTP and C-OXT-GTP were incorporated into DNA and caused chain termination mainly at sites one or two nucleotides beyond the cytosine bases on the template.

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SDS-PAGE showed that human salivary α-amylase family A (HSA-A) was converted to family B (HSA-B) in human saliva. This conversion did not occur in the supernatant of saliva which had been centrifuged at 105, 000×g for 60min. An enzyme which catalyzed the conversion existed in the insoluble fraction of human saliva. The enzyme was solubilized with nonionic or zwitterionic detergents, and showed the maximum activity around pH 6. It was stable between pH 4 and 10, and at a temperature lower than 40°C. The enzyme reduced the molecular weight of HSA-A (62, 000) to the same molecular weight (58, 000) as that of HSA-B without forming any intermediate. It also changed the PAGE pattern of multiple forms of HSA-A to the same pattern as that of HSA-B. It was not inhibited by protease inhibitors, and it did not destroy the reactivity of HSA-A with anti-human salivary α-amylase antiserum. The enzyme diminished the reactivity of HSA-A with concanavalin A. These results indicate that HSA-A was converted to HSA-B through the release of sugar chains by the action of the enzyme in the insoluble fraction of human saliva.

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A 7.6-kb fragment of Salmonella typhimurium LT2 containing the liv gene cluster, which specifies the high-affinity branched-chain amino acid transport system (LIV-I), has been isolated. The upstream region contains the livB and livC genes encoding the leucine-isoleucine-valine-threonine and leucine-specific binding proteins, respectively. In this study, the nucleotide sequence of the 4-kb downstream segment was determined and found to contain four reading frames, designated as livA, livE, livF, and livG, that encode putative membrane-associated proteins. The livA and livE genes encode hydrophobic proteins composed of 308 and 425 amino acid residues, respectively. The livF and livG genes encode hydrophilic proteins of 255 and 237 amino acids, respectively; both the proteins contain consensus amino acid sequences found in proteins with ATP-binding sites. These four genes linked together have a potential rho-independent transcriptional terminator adjacent to the 3'-end of LivG. No promoter sequence was found in the immediate upstream region of the livAEFG cluster. The livA, livE, livF, and livG gene products were identified as proteins with apparent M_rs of 25, 500, 34, 500, 28, 000, and 26, 000, respectively, by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequences of these four proteins showed strong homology to those of the corresponding membrane-associated proteins required for the high-affinity branched-chain amino acid transport systems from both Escherichia coli and Pseudomonas aeruginosa.

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In the previous report, we demonstrated that cAMP negatively regulated α-smooth muscle actin mRNA levels through destabilization of the mRNA [O. Ohara et al. (1991) J. Biochem. 109, 834-839]. We here report that the decline in the α-smooth muscle actin mRNA levels was well correlated with disorganization of microfilaments but not necessarily with the rise in intracellular cAMP levels in rat cultured vascular smooth muscle cells. The decrease in the α-smooth muscle actin mRNA induced by microfilament-disorganizing agents also resulted from enhancement of the turnover rate of the mRNA. These results raise the possibility that the post-transcriptional control of the α-smooth muscle actin expression is linked to the organization of actin filaments in smooth muscle cells.

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Vanadate is known to have various insulin-like actions including activation of D-glucose uptake into the skeletal muscle and adipose tissue. In this study, we examined the effect of orthovanadate on D-glucose uptake into sarcolemmal vesicles prepared from rat hind limb skeletal muscles. In the presence of 10mM vanadate, the initial rate of D-glucose uptake into sarcolemmal vesicles was enhanced 4-5 times above the basal value. Half-maximal concentration for this effect of vanadate was 3mM. The n-glucose uptake was also stimulated by metavanadate, but not by selenite, selenate, or molybdate. When vanadate was removed from the vesicles by dilution and centrifugation, n-glucose uptake into the vesicles returned to the basal level, indicating that the effect of vanadate was reversible. Saturation curves showed that the V_max value for the n-glucose uptake was enhanced more than 4-fold by 10mM vanadate. Therefore, the activation of D-glucose uptake was due, at least in part, to a large increase in the V_max value. These results suggest that vanadate increases the intrinsic activity (turnover number) of skeletal muscle glucose transporters in a reversible manner.

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The regulatory mechanism of cytosolic sulfation of T₃ has been studied in rat liver. Sulfation of T₃ is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 times higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T₃ sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T₃ was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T₃ (50μg/kg daily for 7 days) to thyroidectomized rats. Administration of T₃ in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T₃ sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2, 6-dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T₃ was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human. A sex-related difference was also observed in mice, with the females showing the higher activity.

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Age-related changes in ouabain binding to synaptic plasma membranes isolated from cerebral cortices of C57BL/6 mice were investigated to examine whether the density of Na⁺, K⁺-ATPase decreases with advancing age. Specific binding of [³H]ouabain did not change until around 20 months of age, but a 22% decrease in binding was found in the late senescent stage (29 months). Scatchard analysis of the binding revealed that the maximum number of binding sites (B_max) was lower in aged mice, while the binding affinity (K_d) for ouabain receptor remained unchanged with aging. These results indicate that the density of Na⁺, K⁺-ATPase enzyme sites in the plasma membranes of brain synapses decreases in aged mice. Since the activity of Na⁺, K⁺-ATPase has been found to start declining at a much earlier stage [Tanaka, Y. & Ando, S. (1990) Brain Res. 506, 46-52; Ando, S. & Tanaka, Y. (1990) Gerontology 36, 10-14] than that at which the decrease of B_max is manifested, at least
two mechanisms may underlie the age-related decrease of the enzyme activity. We speculate that the lipid microenvironment which regulates the enzyme activity starts to change at the early stage of senescence, followed by the decrease in the enzyme content in the later stage, that is, both changes cooperatively diminish the Na⁺, K⁺-ATPase activity in senescence.

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A method for preparation of pyridylamino (PA-) derivatives of O-linked sugar chains from glycoproteins was developed. A glycopeptide containing O-linked Galβ1-3GalNAc was prepared from fetuin and treated with anhydrous hydrazine followed by N-acetylation of free amino groups. Sugar chains released were pyridylaminated with improved reaction conditions and excess reagents were removed by gel filtration. Galβ1-3GalNAc-PA obtained together with PA-Gal as a by-product was quantified by HPLC. Conditions for the hydrazine treatment were investigated and the treatment at 40°C for 350h gave the best results for releasing O-linked sugar chains. The total yield of Gal β1-3GalNAc-PA from the glycopeptide was 53% under the established conditions and that of PA-Gal was 18%. The present method was applied to a glycoprotein, and the expected PA-O-linked sugar chains were obtained. Under these conditions, N-linked sugar chains were also released.

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Honeybee α-glucosidase I was inactivated with diethylpyrocarbonate (DEPC). The inactivation followed pseudo-first-order kinetics. The rate of the loss of activity was decreased by the addition of a substrate, maltose. Since there was no spectral change in the tyrosine absorption region, it was recognized that DEPC did not react with this residue. The α-glucosidase had one free sulfhydryl group, which was not involved in the catalytic reaction, and was not modified by DEPC. On the other hand, the specific reaction of DEPC with a histidyl residue was spectrophotometrically confirmed by an increase in absorption near 240nm, and the activity of the inactivated enzyme was restored by hydroxylamine. The modification rate of one histidyl residue by DEPC was almost equal to the rate of the activity loss. These results indicate that there is one histidyl residue at or near the catalytic site, and that honeybee α-glucosidase I has a single active site.

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In order to study the structure-function relationship of an RNase T₂ family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or Hisl09 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the ¹H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pK_a values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pK_a value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the ¹NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.

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Glutamate racemase of Pediococcus pentosaceus contained no cofactor, and was completely inactivated by a thiol reagent. The role of a cysteine residue in the enzyme reaction was studied by chemical modification. The modification of this cysteine residue resulted in a concomitant loss of activity. DL-Glutamate protected the enzyme from inactivation. The inactivated enzyme was reactivated by addition of dithiothreitol. The racemization in ²H₂O showed an overshoot in the optical rotation of glutamate before the substrate was completely racemized. This indicates that the removal of α-hydrogen is the rate determining step. During the racemization of D- or L-glutamate in ³H₂O, tritium was incorporated preferentially into the product. Glutamate is racemized by the enzyme probably through a two base mechanism.

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The interaction of Japanese elderberry bark lectin (Sambucus sieboldiana agglutinin, SSA) with carbohydrate was investigated by ¹H-NMR. When a low affinity ligand, methyl β-D-galactoside (βMeGal), was mixed with SSA, each proton signal of βMeGal was broadened. The signal of H-4 was markedly broad, while those of H-1, OCH₃, and H-2 of βMeGal were rather sharp. The specific broadening of Gal H-4 was more evident when SSA was mixed with methyl-β-D-lactoside (βMeLac). Position-dependent signal broadening suggests that βMeGaI binds to SSA such that H-4 is closely involved in the contact region, but H-1, OCH₃, and H-2 are far from this region. In the case of a high affinity ligand, Neu5Ac(α2-6)Gal(β1-4)Glc(=N6L), ligand signals of the SSA-N6L mixture did not change at all. But when a small amount of N6L was added to the SSA-βMeGal mixture, the broad signals of bound βMeGal became dramatically sharp. This indicates that the added N6L molecules liberated the bound βMeGal from SSA. On the other hand, the sialyllactose with the α(2-3)-linkage(=N3L) could not substitute for bound βMeGal because of its lower affinity. This demonstrates that the competitive binding experiment between two ligands is a useful technique to detect the interaction of lectins with high affinity ligands which could not be observed directly by NMR signal broadening and/or chemical shift change.

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Molecular events associated with the evolution of an enzyme for C₄ photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4. 1. 1. 31] are known: the C₄-form, the C₃-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I. P. & Osmond, C. B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C₃-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C₃-form and C₄-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C₄ -form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C₄-form and C₃-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2×10⁸ yr ago), though the divergence of maize (C₄ plant) from wheat (C₃ plant) is supposed to have occurred much later (6×10⁷ yr ago).

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cDNAs for various chimeras between P450 2C2, P450 2C14, P450 2B5, and P450 2E1 were constructed, the chimeric P450s were expressed in yeast cells, and their catalytic activities were compared in the reconstituted system containing partially purified P450 preparations. The chimera P450 (2Hc3), consisting of the 462 amino-terminal residues of P450 2C2 and the remaining 28 residues of P450 2C14, had testosterone 16β-hydroxylase activity, which is not seen in either of the parental P450s, in addition to higher activities of laurate (ω-1)-hydroxylation and benzphetamine N-demethylation than the parental P450s [Uno, T. et al. (1990) Biochem. Biophys. Res. Commun. 167, 498-503] . When either of the segments from P450 2C2 and P450 2C14 in this chimera was replaced with the corresponding sequences of P450 2E1 or when the 35 carboxy-terminal residues of P450 (2Hc3) were replaced with those of P450 2B5, the 16β-hydroxylase activity disappeared. When the 262 amino-terminal residues, except for residues 90-125 (region 90-125), of P450 (2Hc3) were replaced with those of P450 2C14, the resulting chimera retained both testosterone 16β- and laurate (ω-1)-hydroxylase activities. Further replacing the region 90-125 with that of P450 2C14 resulted in disappearance of the 16β-hydroxylase activity and profound decrease in the (ω-1)-hydroxylase activity. Testosterone 16β-hydroxylation was inhibited by laurate and laurate (ω-1)-hydroxylation by testosterone. These findings indicate that testosterone 16β-hydroxylase activity is produced only when the specific segments of P450 2C2 and P450 2C14 are combined, and that at least the 90-125 region of P450 2C2 participates in both testosterone 16β-hydroxylase and laurate (ω-1)-hydroxylase activities. To analyze the increase in the laurate hydroxylase activity on replacing the carboxy-terminal segment of P450 2C2, the rate of P450 reduction by NADPH and the ratio of NADPH oxidation to the product formation in the reconstituted hydroxylase system were measured. From the results, the possibility is discussed that a local conformational change is induced by replacing the carboxy-terminal segment of P450 2C2 with that of P450 2C14, resulting in improvement of the interaction between the oxygenated P450 intermediate and the electronsupplying system in the rate-limiting step of the overall hydroxylase reaction.

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A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5α. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3. 2. 1. 30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including, β-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3. 2. 1. 52]. The enzyme was resistant to denaturation by 2M NaCl, thermostable at 45°C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.

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Endothelin-1 (ET-1) is a potent peptidic vasoconstrictor. This peptide has been shown to be cleared rapidly by the kidney. The purpose of the present study was to assess the involvement of renal proteolytic enzymes in the clearance/degradation of ET- 1. Incubation of ET-1 with the cytosolic fraction of rat kidney homogenate resulted in a decrease of contractile activity on rabbit aortic rings when compared to the untreated ET-1. This cytosolic fraction was chromatographed by anion-exchange and concanavalin A columns. The partially purified enzyme cleaved off the C-terminal tryptophan of ET-1 rapidly, resulting in a peptide which is three orders of magnitude weaker in potency than ET-1 in causing smooth muscle contraction. In contrast, proendothelin-1 was not degraded by this endothelin degradation enzyme (EDE). The effects of EDE on other vasoactive peptides were also examined. The C-terminal tyrosine of atrial natriuretic peptide was cleaved by EDE, but the biological activity of the resulting peptide was not significantly changed. Angiotensin II was not a substrate for EDE. The EDE was shown to be different from both carboxypeptidases A and B based on the HPLC analysis of the degradation products of ET-1 produced by these enzymes. In addition, these enzymes displayed different sensitivities toward a carboxypeptidase inhibitor from potato tuber. These results suggest that this previously unidentified enzyme inactivates ET-1 effectively and that it may play a role in modulating the levels of ET-1 in the kidney.

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