Substrate Specificity of a Novel Alcohol Resistant Metalloproteinase,Vimelysin, from Vibrio sp. T1800

Vimelysin is a novel alcohol resistant metalloproteinase from Vibrio sp. T1800. The substrate specificity of vimelysin was studied by using natural and furylacryloyl dipeptide substrates. Vimelysin cleaved mainly Pro⁷-Phe⁸ bond and slightly Tyr⁴-Ile⁵ bond in human angiotensin I. Vimelysin also cleaved mainly Phe²⁴-Phe²⁵ and Tyr¹⁶-Leu¹⁷ bonds, and slightly His⁵-Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴-Leu¹⁵, and Gly²³-Phe²⁴ bonds in oxidized insulin B-chain. The substrate specificity of vimelysin, by using furylacryloyl (Fua) dipeptides were also studied. The ratio of k_cat/K_m for Fua-Gly-Phe-NH₂/Fua-Gly-Leu-NH₂, Fua-Phe-Leu-NH₂/Fua-Gly-Leu-NH₂, and Fua-Phe-Phe-NH₂/Fua-Gly-Leu-NH₂ were 15.9, 27.8, and 59.0, respectively. These results indicate that vimelysin easily recognizes phenylalanine in P1′ positions, which is different from thermolysin.     

Purification,characterization and substrate specificity of a basic proteinase in the venom of Habu (Trimeresurus flavoriridis)

A basic proteinase was purified and characterized from the venom of Habu (Trimeresurus flavoviridis). Its molecular weight, isoelectric point and optimum pH were approx. 24 000, 9.2 and 9, respectively. Susceptibility to several reagents was examined. The proteinase had endopeptidase activity cleaving the Gly-Leu bond in synthetic peptides but no exopeptidase activity. It did not hydrolyze a peptide, Z-Gly-Pro-Leu-Gly-Pro, which had been a good substrate for the major proteinase in the venom. The proteinase cleaved oxidized insulin B chain at five positions: His₁₀-Leu₁₁, Ala₁₄-Leu₁₅, Tyr₁₆-Leu₁₇, Gly₂₃-Phe₂₄ and Phe₂₄-Phe₂₅. From the disappearance of intermediate peptides and the peptides accumulated, the order and the intensity of cleavage of these positions were determined, and the substrate specificity was compared with those hitherto described for hemorrhagic and nonhemorrhagic venom proteinases.     

Substrate specificity of carboxyl proteinase fromPycnoporus coccineus,a wood-deteriorating fungus

A carboxyl proteinase was purified from submerged-culture filtrate of a wood-deteriorating basidiomycete,Pycnoporus coccineus. The purified enzyme was found to be essentially homogeneous in disc gel electrophoresis tests at pH 9.4 and 2.3. The specificity and mode of action ofP. coccineus carboxyl proteinase I_a were investigated with the oxidized B-chain of insulinP. coccineus carboxyl proteinase I_a hydrolyzed primarily three peptide bonds, Ala¹⁴-Leu¹⁵, Tyr¹⁶-Leu¹⁷, and Phe²⁴-Phe²⁵ bonds, in the oxidized B-chain of insulin.     

D-isomeric replacements within the 6-9 core sequence of Ac-[Nle4]-alpha-MSH4-11-NH2: a topological model for the solution conformation of alpha-melanotropin

Analogs of Ac-[Nle⁴]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷]-α-MSH_4–11-NH₂ were prepared with D -isomeric replacements at the His⁶, Arg⁸, and Trp⁹ residues. The requirement for an indole moiety at position 9 also was evaluated by replacement with L -leucine in both parent fragment analogs. D -isomeric replacements at positions 6 and 8 in either series were detrimental to biological potency in frog (Rana pipiens) and lizard skin (Anolis carolinensis) in vitro melanotropic assays. However, Ac-[Nle⁴, D -Trp⁹]-α-MSH_4–11-NH₂ and Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ were equipotent and 10 × more potent than Ac-[Nle⁴]-α-MSH_4–11-NH₂, respectively, in the lizard skin bioassay, and 30 and 1900 times more potent in the frog skin bioassay. Ac-[Nle⁴, D -Phe⁷, D -Trp⁹]-α-MSH_4–11-NH₂ was 3 × more potent than α-MSH in the frog skin bioassay. Proton nmr studies in aqueous solution revealed a marked preservation of the backbone conformation of these linear analogs. Chemical-shift variations due to the through-space anisotropic influence of the core aromatic amino acid residues permitted evaluation of side-chain topology. The observed topology was consistent with nonhydrogen-bonded β-like structure (? = ?139°, ψ = +135° for L -amino acids; ? = +139°, ψ = ?135° for D -amino acids) as the predominant solution conformation. The biological and conformational data suggest that high melanotropic potency requires a close spatial arrangement of the His⁶, Phe⁷, and Arg⁸ side chains.     

Substrate specificity of carboxyl proteinase ofAspergillus sojae

The specificity and mode of action ofAspergillus sojae carboxyl proteinase I were investigated with the oxidized B-chain of insulin.A. sojae carboxyl proteinase I hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu¹⁵-Tyr¹⁶ bond and the Phe²⁴-Phe²⁵ bond. Additional cleavage of the bond Tyr¹⁶-Leu¹⁷ was also noted.     

Carboxyl Proteinase from the Wood Deteriorating Basidiomycete Pycnoporus coccineus :Substrate Specificity with Oxidized Insulin Peptide B1 ~ B16 and B15 ~ B24, Angiotensin and Proangiotensin

The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr⁴-Ile⁵ bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′₁ position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus.     

Conformational energy studies on N-methylated analogs of thyrotropin releasing hormone, enkephalin, and luteinizing hormone-releasing hormone

Empirical conformational energy calculations have been carried out for N-methyl derivatives of alanine and phenylalanine dipeptide models and N-methyl-substituted active analogs of three biologically active peptides, namely thyrotropin-releasing hormone (TRH), enkephalin (ENK), and luteinizing hormone-releasing hormone (LHRH). The isoenergetic contour maps and the local dipeptide minima obtained, when the peptide bond (ω) preceding the N-methylated residue is in the trans configuration show that (1) N-methylation constricts the conformational freedom of both the ith and (i + 1)th residues; (2), the lowest energy position for both residues occurs around ? = ?135° ± 5° and ψ = 75° ± 5°, and (3) the α_L conformational state is the second lowest energy state for the (i + 1)th residue, whereas for the ith residue the C₅ (extended) conformation is second lowest in energy. When the peptide bond (ω_i) is in the cis configuration the ith residue is energetically forbidden in the range ? = 0° to 180° and ψ = ?180° to +180°. Conformations of low energy for ω_i = 0° are found to be similar to those obtained for the trans peptide bond. In all the model systems (irrespective of cis or trans), the α_R conformational state is energetically very high. Significant deviations from planarity are found for the peptide bond when the amide hydrogen is replaced by a methyl group. Two low-energy conformers are found for [(N-Me)His²]TRH. These conformers differ only in the ? and ψ values at the (N-Me)His² residue. Among the different low-energy conformers found for each of the ENK analogs [D -Ala²,(N-Me)Phe⁴, Met⁵]ENK amide and [D -Ala²,(N-Me)Met⁵]ENK amide, one low-energy conformer was found to be common for both analogs with respect to the side-chain orientations. The stability of the low-energy structures is discussed in the light of the activity of other analogs. Two low-energy conformers were found for [(N-Me)Leu⁷]LHRH. These conformations differ in the types of bend around the positions 6 and 7 of LHRH. One bend type is eliminated when the active analog [D -Ala⁶,(M-Me)Leu⁷]LHRH is considered.     

Existence of Met-enkephalin-Arg6-Gly7-Leu8 with Met-enkephalin,Leu-enkephalin and Met-enkephalin-Arg6-Phe7 in the brain of guinea pig,rat and golden hamster

High performance liquid chromatography (HPLC) coupled with specific radioimmunoassays for methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸ (Met-E-Arg⁶-Gly⁷-Leu⁸), methionine-enkephalin (Met-E), leucine-enkephalin (Leu-E) and methionine-enkephalin-Arg⁶-Phe⁷ (Met-E-Arg⁶-Phe⁷) has demonstrated that Met-E-Arg⁶-Gly⁷-Leu⁸ exists together with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ in the brain of guinea pig, rat and golden hamster. The content of Met-E-Arg⁶-Gly⁷-Leu⁸ was comparable to those of Leu-E and Met-E-Arg⁶-Phe⁷, whereas that of Met-E was the highest among the four opioid peptides. These results are compatible with the recent studies on the nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla, which reveal that this precursor molecule contains four copies of Met-E and one copy each of Leu-E, Met-E-Arg⁶-Phe⁷ and Met-E-Arg⁶-Gly⁷-Leu⁸. The co-existence of Met-E-Arg⁶-Gly⁷-Leu⁸ with Met-E, Leu-E and Met-E-Arg⁶-Phe⁷ suggests that their biosynthetic pathway in the brain is similar to that in the adrenal medulla.     

Gene cloning,expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacterium Psychrobacter sp. C18

A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence LipX was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET28a(+)/lipX gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipX_His with a 6× histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30°C and pH 8.0 with p-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipX_His is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn²⁺, Co²⁺, Mn²⁺, Fe³⁺ and EDTA. Most non-ionic detergents, such as DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Additionally, the highest lipolytic rate of the recombinant LipX_His lipase was achieved when p-nitrophenyl myristate was used as a substrate, among all the p-nitrophenyl esters tested.     

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His₆-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg⁻¹ protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His₆-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co²⁺ and Mn²⁺ ions. His₆-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k_cat/K_m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s⁻¹ mM⁻¹, respectively.     

Interaction between poly(L-lysine48, L-histidine52) and DNA.

Poly(Lys⁴⁸, His⁵²), a random copolypeptide of L -lysine (48%) and L -histidine (52%), was used as a model protein for investigating the effects of protonation on the imidazole group of histidines on protein binding to DNA. The complexes formed between poly(Lys⁴⁸, His⁵²) and DNA were examined using absorbance, circular dichroism (CD), and thermal denaturation. Although increasing pH reduces the charges on histidine side chains in the model protein, the protein still binds the DNA with approximately one positive charge per negative charge in protein-bound regions. Nevertheless, CD and melting properties of poly(Lys⁴⁸, His⁵²)-DNA complexes still depend upon the solution pH which determines the protonation state of imidazole group of histidine side chains. At pH 7.0, the complexes show two characteristic melting bands with a t_m (46–51°C) for free base pairs and a t′_m (94°C) for protein-bound base pairs. The t′_m of the complexes is reduced to 90°C at pH 9.2, although at this pH there is still one lysine per phosphate in protein-bound regions. Presumably, the presence of deprotonated histidine residues destabilizes the native structure of protein-bound DNA. The binding of this model protein to DNA causes a red shift of the crossover point and both a red shift and a reduction of the positive CD band of DNA near 275 nm. This phenomenon is similar to that caused by polylysine binding. These effects, however, are greatly diminished when histidine side chains in the model protein are deprotonated. The structure of already formed poly(Lys⁴⁸, His⁵²)·DNA complexes can be perturbed by changing the solution pH. However, the results suggest a readjustment of the complex to accommodate charge interactions rather than a full dissociation of the complex followed by reassociation between the model protein and DNA.     

Characterization of the activation of pro-urokinase by thermolysin

The bacterial metalloproteinase thermolysin catalyzes the efficient activation of pro-urokinase to an active high-molecular-weight form of the protein. Thermolysin and plasmin convert pro-urokinase to enzymes of essentially equal activities in amidolytic assays, but with different molecular structures. The B-chains of the proteins produced by thermolysin and plasmin are of the same size (33 kDa) and have the same amino-terminal sequences, demonstrating that the cleavage of the Lys¹⁵⁸-Ile¹⁵⁹ bond of pro-urokinase is catalyzed by both enzymes. However, thermolysin also reacts at additional sites in the growth factor domain of the A-chain at nearly the same rate as that of the activation reaction. Polypeptides derived from hydrolyses of the Glu³-Leu⁴, Tyr²⁴-Phe²⁵, Asn²⁷-Ile²⁸ and Lys³⁶-Phe³⁷ bonds are recovered after reduction of the activated protein. The carboxy-terminus of the A-chain has been shown to be Arg-156, a consequence of proteolysis of the Arg¹⁵⁶-Phe¹⁵⁷ bond. In contrast to plasmin, thermolysin activates thrombin-inactivated pro-urokinase nearly as rapidly as it does the native zymogen. Thermolysin provides a useful alternative to plasmin for the catalytic activation and analysis of pro-urokinase, since the bacterial metalloproteinase is stable in solution and not susceptible to inhibition by aprotinin and other serine proteinase inhibitors.     

Specificity of Thermophilic Streptomyces Alkaline Proteinase

The specificity of highly purified alkaline proteinase B (EC 3.4.21.14) from thermophilic Streptomyces rectus var. proteolyticus was investigated with an oxidized insulin B chain. Hydrolysis of the oxidized insulin B chain in a 4-hr incubation was observed mainly at three peptide bonds (Phe²⁴-Phe²⁵, Leu¹⁵-Tyr¹⁶ and Leu¹¹-Val¹²) and additionally at six others (Leu⁶-CySO₃H⁷, Gln⁴-His⁵, Leu¹⁷-Val¹⁸, His⁵-Leu⁶, Glu¹³-Ala¹⁴, Asn³-Gln⁴).

Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotension (formerly designated angiotensin I) was observed at the Tyr⁴-Ile⁵ and Phe⁸-His⁹ bonds.     

Specificities of extracellular and ribosomal serine proteinases from Bacillus natto,a food microorganism

The specificities of extracellular and ribosomal serine proteinase from Bacillus natto, a food microorganism, were investigated. Both proteins have highly restricted and characteristic specificities. With the extracellular serine proteinase, initial cleavage site was observed at Leu¹⁵-Tyr¹⁶, secondary site at Ser⁹-His¹⁰ and additional cleavage sites at Gln⁴-His⁵ and His⁵-Leu⁶ in the oxidized insulin B-chain. Hydrolysis of proangiotensin with the extracellular serine proteinase was observed primarily at Phe⁸-His⁹ and secondary at Tyr⁴-Ile⁵. The extracellular serine proteinase has a K_m of 0.08 mM and k_cat of 3 s⁻¹ for angiotensin hydrolysis. With the ribosomal proteinase, initial cleavage site of the oxidized insulin B-chain was observed at Leu¹⁵-Tyr¹⁶ and additional cleavage site at Phe²⁴-Phe²⁵. Hydrolysis of proangiotensin was observed at Tyr⁴-Ile⁵ bond with the ribosomal proteinase.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K _m of 10.55 ± 0.9 μM, a k _cat of 0.6 ± 0.01 s⁻¹ and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAα1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.     

The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme

To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His₆-tagged EcDCP (His₆-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity by Ni²⁺-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His₆-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His₆-EcDCP was stimulated by 1.5 fold. The K _M and k _cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s⁻¹, respectively. His₆-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His₆-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17% α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme.     

Nmr and computer-aided studies of the three interchanging stereoisomers of the cyclic hexapeptide cyclo[-Pro1-Gly2-Glu3(OBzl)-Pro4-Phe5-Leu6-]: Unexpected observation of a cis isomer of a secondary amide peptide bond in the presence of two trans proline peptide bonds

The cyclic hexapeptide cyclo[-Pro¹-Gly²-Glu³(OBzl)-Pro⁴-Phe⁵-Leu⁶-] ( 1 ; OBzl: benzyl ester) was modeled and synthesized to be used as a chiral site for the separation of enantiomers. Total correlation spectroscopy and nuclear Ovehauser effect spectroscopy spectra of the peptide in CDCl₃ showed the presence of three stereoisomers. The two dominant stereoisomers 1a and 1b exchanged chemically with each other, while the minor stereoisomer 1c exchanged exclusively with the stereoisomer 1b . Stereoisomer 1a had two cis proline peptide bonds while stereoisomer 1b had all-trans peptide bonds. The stereoisomer 1c had, for nonstrained peptides, an unusual cis phenylalanine peptide bond while both proline peptide bonds were trans. © 1993 John Wiley & Sons, Inc.     

Purification and Characterization of N-Acetylglucosamine-6-phosphate Deacetylase from a Psychrotrophic Marine Bacterium, Alteromonas Species

A psychrotrophic bacterium, strain Mct-9, which produced an N-acetylglucosamine-6-phosphate deacetylase, was isolated from a deep-seawater sample in the Mariana Trough. The Mct-9 strain was identified as Alteromonas sp. The native enzyme had a molecular mass of 164,000 Da, and was predicted to be composed of four identical subunits with molecular masses of 41,000 Da. The purified enzyme hydrolyzed N-acetylglucosamine (GlcNAc), GlcNAc-6-phosphate, and GlcNAc-6-sulfate. Considering the low K _m and high k _cat /K _m for GlcNAc-6-phosphate, it probably acts as a GlcNAc-6-phosphate deacetylase in vivo. The enzyme was functional in the temperature range of 5° to 70°C and displayed optimal activity at 55°C. The optimal temperature was higher than that of the deacetylase from the mesophilic bacterium Vibrio cholerae non-O1. The characteristics of the GlcNAc-6-phosphate deacetylase from Alteromonas sp. are unique among psychrotrophs and psychrophiles, whose intracellular enzymes are mostly thermolabile. Received May 6, 1999; accepted August 16, 1999.     

The novel metalloproteinase atroxlysin-I from Peruvian Bothrops atrox (Jergón) snake venom acts both on blood vessel ECM and platelets

We report the isolation and structure-function relationship of a 23 kDa metalloproteinase named atroxlysin-I from the venom of the Peruvian Bothrops atrox (Jergón). Atroxlysin is a P-I metalloproteinase and contains 204 residues. Its proteolytic activity towards dimethylcasein is enhanced by Ca⁺² but inhibited by EDTA, dithiothreitol, excessive Zn⁺² and α2-macroglobulin. Unlike other structurally homologous P-I metalloproteinases, atroxlysin-I causes hemorrhages. To examine its hemorrhagic activity mechanistically, we studied its function in vitro and in vivo. It cleaved the Ala₁₄-Leu₁₅ and Tyr₁₆-Leu₁₇ bonds in oxidized insulin B-chain and specifically hydrolyzed the α-chains of fibrin(ogen) in a dose- and time-dependent manner. Atroxlysin-I cleaved plasma fibronectin and other extracellular matrix proteins (collagens I and IV) and the triple-helical fragment CB3 of collagen IV, but did not degrade laminin-111. Complementarily, the laminin and collagen binding integrins α₇β₁ and α₁β₁ were cleaved by atroxlysin. Even without catalytic activity atroxlysin-I inhibited collagen- and ADP-triggered platelet aggregation.