Coesite and stishovite in a shocked lunar meteorite, Asuka-881757, and impact events in lunar surface.

Microcrystals of coesite and stishovite were discovered as inclusions in amorphous silica grains in shocked melt pockets of a lunar meteorite Asuka-881757 by micro-Raman spectrometry, scanning electron microscopy, electron back-scatter diffraction, and transmission electron microscopy. These high-pressure polymorphs of SiO(2) in amorphous silica indicate that the meteorite experienced an equilibrium shock-pressure of at least 8-30 GPa. Secondary quartz grains are also observed in separate amorphous silica grains in the meteorite. The estimated age reported by the (39)Ar/(40)Ar chronology indicates that the source basalt of this meteorite was impacted at 3,800 Ma ago, time of lunar cataclysm; i.e., the heavy bombardment in the lunar surface. Observation of coesite and stishovite formed in the lunar breccias suggests that high-pressure impact metamorphism and formation of high-pressure minerals are common phenomena in brecciated lunar surface altered by the heavy meteoritic bombardment.

Hanging drop monoculture for selection of optimal antioxidants during in vitro maturation of porcine oocytes.

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre-culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 µg/ml of L-carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non-presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 µg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.

Defective glycine cleavage system in nonketotic hyperglycinemia. Occurrence of a less active glycine decarboxylase and an abnormal aminomethyl carrier protein.

The activities of then glycine cleavage system in the liver and brain of patient with nonketotic hyperglycinemia was extremely low as compared with those of control human liver and brain. The activities of glycine decarboxylase (P-protein) and the aminomethyl carrier protein (H-protein), two of the four protein components of the glycine cleavage system, were considerably reduced in both the liver and brain; the extent of reduction was greater in the H-protein. The activity of the T-protein was normal. Purified H-protein from the patient did not react with lipoamide dehydrogenase, and titration of thiol groups with [2,3-14C]N-ethylmaleimide suggested that this H-protein is devoid of lipoic acid. This structural abnormality in the H-protein is considered to constitute the primary molecular lesion in this patient with non-ketotic hyperglycinemia. Immunochemical studies using an antibody specific for P-protein showed that the patient was due to reduction of the catalytic activity of the protein rather than a decrease in the actual amount of the P-protein. Partial inactivation of P-protein could result secondarily from impaired metabolism of glycine resulting from deficiency in the activity of H-protein. However, the H-protein from the patient could stimulate the P-protein catalyzed exchange of the carboxyl carbon of glycine with 14CO2, although the specific activity of the purified H-protein from the patient was only 4% of that of control human H-protein. The content of H-protein in the liver of the patient was approximately 35% of that of control human liver.

Identification and characterization of nuclear calmodulin-binding proteins of Saccharomyces cerevisiae.

Nuclear calmodulin-binding proteins of the yeast Saccharomyces cerevisiae were investigated. The soluble fractions after serial treatments of the isolated nuclei with buffers containing the nonionic detergent NP-40 (F1), 0.5 M KCl (F2) and 2.0 M KCl (F3) in this order, and the residual proteins (F4) were obtained. The calmodulin-binding proteins of the nucleus and nuclear subfractions were identified using the gel overlay method using 125I-calmodulin. Each subnuclear fraction contained a large number of components that bound calmodulin in a Ca(2+)-dependent or -independent manners. The calmodulin-binding proteins were isolated from F1 and F2 subnuclear fractions by affinity chromatography. The affinity-purified proteins bound calmodulin in a Ca(2+)-dependent manner when analyzed using the gel overlay method. The major calmodulin-binding components of F1 were 44, 42, 36, 32 and 29 kDa proteins, and those of F2 were 200, 100, 40, 42, 36, 34 and 32 kDa proteins. The isolated proteins also contained several Coomassie-blue stained proteins that did not bind calmodulin and, therefore, may represent the proteins associated with the calmodulin-binding proteins. Antisera raised against the affinity-purified preparation of F1 and F2 recognized almost all of the calmodulin-binding proteins present in the fraction and several other proteins of the nucleus. The presence of Ca(2+)-dependent protein phosphatase (type 2B) in the nucleus was demonstrated by Western blotting. The enzyme was localized predominantly in F1 and F4.

Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8.

We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.

NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral beta gamma-crystallin precursor structure.

The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Calpha, C') r.m.s.d. values of 0. 45(+/-0.11) A and 0.57(+/-0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two beta-sheets, each consisting of four antiparallel beta-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved.

Elucidation of the mode of interaction of thermolysin with a proteinaceous metalloproteinase inhibitor, SMPI, based on a model complex structure and a structural dynamics analysis.

SMPI is a proteinaceous microbial metalloproteinase inhibitor that was isolated from Streptomyces nigrescens TK-23 in 1979. SMPI is known to selectively inhibit the metalloproteinases in the gluzincin family, according to the Rawling and Barrett classification. There has been no report on the interaction of a metalloproteinase in the family of gluzincins with its specific proteinaceous inhibitor. We have solved the solution structure of SMPI by NMR. Here, we report the binding mode of SMPI to thermolysin, based on the model complex structure generated using our high-resolution NMR structure of SMPI and the crystal structure of thermolysin. The obtained complex model shows that the extruded loop of SMPI, with the scissile bond Cys64-Val65, is complementary in shape to the active cleft of thermolysin. In the complex, the Cys64 (P1) carbonyl oxygen atom can form a tetrahedral coordination to the active zinc in thermolysin, and simultaneously, the methyl groups of Val65 (P1') are closely located in the hydrophobic S1' pocket in thermolysin. From the electrostatic potential surface calculation, the active loop of SMPI and the active cleft in thermolysin have been shown to be complementary in the surface charge distribution, resulting in the stabilization of the complex. The apparently large active loop is less flexible, but maintains a conformation in the nano- to picosecond time-scale, as elucidated from the 15N spin relaxation analysis. This is a quite different structural feature of SMPI from the flexible binding loop generally found in the serine proteinase inhibitors, such as SSI and eglin c, and can be related to the narrow specificity of SMPI. The present study provides the first insight into the interaction between a proteinaceous inhibitor and a gluzincin metalloproteinase.

NMR structure of Streptomyces killer toxin-like protein, SKLP: further evidence for the wide distribution of single-domain betagamma-crystallin superfamily proteins.

A protein isolated from the culture supernatant of the soil bacterium, Streptomyces sp. F-287, exhibits cytocidal effects for both budding and fission yeasts, and causes morphological changes of yeasts and filamentous fungi. This protein, which was the first killer toxin-like protein for yeasts identified in the Streptomyces microorganism, was named SKLP (Streptomyces killer toxin-like protein). Since the amino acid sequence of the protein, as determined by sequential Edman degradations, seemed to be unique, we determined the structure by NMR spectroscopy. Although the actual target of SKLP in yeasts has not been determined yet, the structure might give us a clue to characterize the targets. The solution structure of SKLP determined by NMR, however, turned out to be a single-domain crystallin-like protein, with two Greek key motifs and a short extra beta-strand at the N terminus. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with rmsd values of 0.32(+/-0.06) A for the backbone atoms involved in the secondary structure elements. As a yeast killer toxin, WmKT, isolated from the yeast strain Williopsis mrakii also has a Greek key beta-barrel fold, we have made a detailed comparison of the structural features of SKLP with the other crystallin superfamily proteins. It is very interesting that SKLP has a unique electrostatic potential distribution on the molecular surface. Namely, one surface of the beta-barrel fold in SKLP has a large negatively charged region, with an isolated positive charge of the Arg62 side-chain at the center. The edge of this surface is surrounded by positively charged residues, including Arg31, Arg65 and Arg74. The salient features of the charge distribution on this surface and the cluster of Arg residues might be related to the target binding of SKLP.

Identification of a wheat germ agglutinin-sensitive ATPase in yeast nuclei.

We have found that wheat germ agglutinin (WGA), a lectin that specifically binds to N-acetylglucosamine residues inhibits the in vitro transport of plasmid DNA, pJDB219, into yeast nuclei. Histochemical staining of the isolated nuclei with biotinylated WGA and streptavidin-biotinylated peroxidase complex revealed the presence of WGA-binding materials around the nuclear pore under an electron microscope. Using WGA-agarose column chromatography of yeast nuclear extracts, a novel Mg2+-dependent ATPase was isolated. Its activity was highly sensitive to WGA and stimulated by Nonidet P-40 or phosphatidylserine. We suggest that the WGA-sensitive ATPase plays a role in yeast nuclear transport of DNA.

Cloning and characterization of the elongation factor EF-1 beta homologue of Saccharomyces cerevisiae. EF-1 beta is essential for growth.

A Saccharomyces cerevisiae cDNA homologue of the elongation factor EF-1 beta was found among the clones obtained by immunoscreening of a yeast cDNA expression library with an antibody against calmodulin affinity-purified proteins. The cDNA encoded a protein of 206 amino acids which was highly homologous (about 70% homology) with Artemia salina and human EF-1 beta. A protein with an apparent molecular mass of 33,000, significantly larger than that expected from the gene, was identified by Western blotting. Gene disruption experiments indicated that EF-1 beta is essential for growth.

Tubulin and high molecular weight microtubule-associated proteins as endogenous substrates for protein carboxymethyltransferase in brain.

The endogenous substrate for protein carboxymethyltransferase in brain was examined. Several polypeptides were methylated when brain slices were incubated with L-methionine or when subcellular fractions of brain, such as the cytosolic fraction, were incubated with S-adenosyl L-methionine. Two methyl-accepting proteins in the cytoplasm were identified as tubulin and high molecular weight microtubule-associated proteins (300 kDa), which are components of microtubules. Tubulin behaved as a 43 kDa protein in acidic polyacrylamide gel electrophoresis, but as a 55 kDa protein in SDS-polyacrylamide gel electrophoresis. The methyl moiety transferred to these proteins from L-methionine was labile at alkaline pH. The high molecular weight microtubule-associated proteins showed higher methyl-accepting activity than tubulin or ovalbumin, which was used as a standard substrate: about 20 mmol of high molecular weight microtubule-associated proteins, 2 mmol of tubulin and 10 mmol of ovalbumin were methylated per mol of each protein in 30 min under the experimental conditions used.

On the mechanism of covalent binding of butylated hydroxytoluene to microsomal protein.

The structures of cysteine conjugates of 3,5-di-tert-butyl-4-hydroxytoluene (BHT) and the binding sites of BHT metabolites on microsomal protein were investigated by 13C nuclear magnetic resonance (13C-NMR) and gas-liquid chromatography/mass spectrometry. The cysteine conjugates of 2,6-di-tert-butyl-4-hydroxymethylphenol (BHT-alcohol) and 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (quinone methide), which are metabolites of BHT found in rat liver and specifically reacts with thiol compounds, were prepared as alcoholic aqueous solutions. The molecular structure of the cysteine conjugate of BHT-alcohol agreed completely with that of quinone methide in 13C-NMR spectra or mass spectra. These spectra of both conjugates further showed that the conjugates are due to thioether binding between the 4-methyl group of metabolites and the sulfhydryl group of cysteine. When [14C]BHT-bound microsomes prepared in vitro were enzymatically hydrolyzed with Pronase E, the major radioactive material that eluted with methanol from a column of Amberlite XAD-2 and gave a positive ninhydrin reaction was identified as a cysteine conjugate of BHT by comparing its Rf values on TLC and mass spectrum. On the basis of the results, it was apparent that the binding site of activated substituents of BHT on protein was mainly the sulfhydryl group of cysteine residue.

On the mechanism of butylated hydroxytoluene-induced hepatic toxicity in rats.

The relations between serum transaminase activity and the hepatic contents of glutathione and lipid peroxide were examined following oral administration to rats of butylated hydroxytoluene (BHT; 500 or 1000 mg/kg). The glutathione level rapidly diminished and reached a minimum at 6 hr after BHT administration. The period of depletion was dependent on dose: restoration of the glutathione level took longer in high-dose rats than in low-dose rats. The content of hepatic lipid peroxide was not markedly changed by BHT throughout the experimental period. The activity of glutathione S-transferase was not affected until 12 hr after BHT administration but, thereafter, it increased with time and was accompanied by elevation of the glutathione level. Though the activities of serum glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase were not affected by low-dose BHT, they increased rapidly in the high-dose rates after a lag period of about 6 hr and reached a maximum at 24 hr after administration; at that time, the livers of the high-dose rats showed centrilobular necrosis. The results indicate that acute hepatic injury was induced by the high-dose BHT. Pretreatment with cobaltous chloride inhibited the increase in the activities of the serum transaminases produced by the high-dose of BHT accompanying the depletion of microsomal cytochrome P-450 content and the induction of glutathione content. These observations suggest that hepatic damage was associated with prolonged depletion of glutathione rather than with lipid peroxidation in the liver, and that the activated metabolites of BHT rather than the parent compound induced the tissue damage.

The mitochondrial glycine cleavage system: differential inhibition by divalent cations of glycine synthesis and glycine decarboxylation in the glycine-CO2 exchange.

The exchange of glycine carboxyl carbon with CO2 catalyzed by the combination of chicken liver glycine decarboxylase (P-protein) and aminomethyl carrier protein (H-protein) was markedly inhibited by various divalent cations, although extents of inhibition by individual metal ions varied considerably. Cu2+ and Zn2+, at 100 microM, inhibited the reaction almost completely, and the inhibitions by Co2+ and Ni2+ were also significant, while Mg2+ and Mn2+ did not appreciably affect the reaction. The inhibition by Zn2+ was competitive with both bicarbonate and H-protein and non-competitive with glycine. Of the two reactions involved in the glycine-CO2 exchange, decarboxylation of glycine yielding the H-protein-bound aminomethyl moiety was not significantly affected by 100 microM Zn2+ or Cu2+, but carboxylation of the H-protein-bound aminomethyl moiety to form glycine was strongly inhibited by either Zn2+ or Cu2+. Various degrees of inhibition of the glycine-CO2 exchange by other divalent metal ions could also be accounted for by the inhibition of the carboxylation step of the exchange reaction. The primary site of the action of divalent metal ions is likely to be not P-protein but H-protein, and the binding of metal ions with the H-protein-bound intermediate of glycine decarboxylation was assumed to account for the observed marked inhibition.

The mitochondrial glycine cleavage system: inactivation of glycine decarboxylase as a side reaction of the glycine decarboxylation in the presence of aminomethyl carrier protein.

Glycine decarboxylase, tentatively called P-protein, was inactivated when it was incubated with glycine in the presence of the aminomethyl carrier protein, called H-protein. The inactivation was accompanied by a spectral change in the P-protein as a pyridoxal phosphate enzyme; the spectrum became unusual, with a peak at 330 nm. The fluorescence emission spectrum of the inactivated P-protein showed a distinct peak at 390 nm when excited at 325 nm. Such a spectral change and concomitant inactivation of the P-protein could be completely prevented by the addition of sodium bicarbonate, which initiates the glycine-CO2 exchange in the reaction mixture. The inactivated P-protein was associated with H-protein and the methylene carbon of glycine, but not the carboxyl carbon, in a manner not separable by gel filtration, although the molar ratios of those three components were not constant. The H-protein recovered in the inactivated P-protein fraction was also catalytically inactive. Neither the pyridoxal derivative nor the methylene carbon of glycine appeared to be covalently bound with the protein, and the methylene carbon could be recovered as formaldemethone when treated with dimedone. The inactivation of the P-protein appears to represent a suicide reaction of the P-protein as a side reaction of the glycine decarboxylation, which is supposed to involve the formation of a ternary complex of P-protein, aminomethyl moiety of glycine and H-protein through a Schiff base linkage of the H-protein-bound amino-methyl moiety with the pyridoxal phosphate of P-protein.

Intracellular distribution of fumarase in various animals.

The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.

Resynthesis of reactive site peptide bond and temporary inhibition of Streptomyces metalloproteinase inhibitor.

Streptomyces metalloproteinase inhibitor (SMPI) is a small proteinaceous inhibitor which inhibits metalloproteinases such as thermolysin (Ki =1.14 x 10(-10) M). When incubated with the enzyme, it is gradually hydrolyzed at the Cys64-Val65 peptide bond, which was identified as the reactive site by mutational analysis. To achieve a further understanding of the inhibition mechanism, we attempted to resynthesize the cleaved reactive site by using the enzyme catalytic action. The native inhibitor was resynthesized from the modified inhibitor (Ki =2.18 x 10(-8) M) by incubation with a catalytic amount of thermolysin under the same conditions as used for hydrolysis (pH 7.5, 25 degrees C), suggesting that SMPI follows the standard mechanism of inhibition of serine proteinase inhibitors. Temporary inhibition was observed when the native inhibitor and thermolysin were incubated at a 1:100 (mol/mol) enzyme-inhibitor ratio at 37 degrees C. SMPI showed temporary inhibition towards all the enzymes it inhibited. The inhibitory spectrum of SMPI was analyzed with various metalloproteinases based on the Ki values and limited proteolysis patterns. Pseudomonas elastase and Streptomyces griseus metalloproteinase II formed more stable complexes and showed much lower Ki values (approximately 2 pM) than thermolysin. In the limited proteolysis experiments weak inhibitors were degraded by the enzymes. SMPI did not inhibit almelysin, Streptomyces caespitosus neutral proteinase or matrix metalloproteinases. SMPI specifically inhibits metalloproteinases which are sensitive to phosphoramidon.

Purification and properties of glycine decarboxylase, a component of the glycine cleavage system, from rat liver mitochondria and immunochemical comparison of this enzyme from various sources.

Glycine decarboxylase, tentatively called P-protein as a constituent of the glycine cleavage system, was purified to near homogeneity from rat liver mitochondria. The purified P-protein was a homodimer with a molecular weight of about 210,000, consisting of identical subunits with a molecular weight of 105,000. In the exchange reaction of the carboxyl carbon of glycine wih CO2 catalyzed by the purified P-protein in the presence of H-protein, the pH optimum was 6.7, Km for glycine was 6.6 mM, and Km for H-protein was 7.4 microM. A specific rabbit antibody against the purified rat liver P-protein was prepared. Ouchterlony double diffusion analysis and immunoinhibition experiments using this antibody revealed immunological cross-reactivity among the P-proteins from various species of animals such as carp, frog, snake, chicken, bovine, and human, suggesting a quite conservative evolution of the glycine cleavage system.

Identification of reactive site of a proteinaceous metalloproteinase inhibitor from Streptomyces nigrescens TK-23.

Streptomyces metalloproteinase inhibitor (SMPI), isolated from Streptomyces nigrescens TK-23, is a small proteinaceous metalloproteinase inhibitor consisting of 102 amino acid residues and two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. After prolonged incubation with a catalytic amount of thermolysin, it is cleaved at Cys64-Val65 [Murai, H., Hara, S., Ikenaka, T., Oda, K., and Murao, S. (1985) J. Biochem. 97, 173-180]. Hence, for identification of the reactive site, mutants were constructed by substituting Val65 with various amino acid residues (Leu, Ile, Phe, Tyr, Gly, Ser, Lys, and Glu). The mutants were analyzed for inhibitory activity. Among them, V65I, V65L, V65F, and V65Y retained strong inhibitory activity, whereas V65S, V65G, V65K, and V65E showed very weak inhibitory activity against thermolysin. The Ki values were found to be of the order of 10(10) M by using a fluorogenic substrate, MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. In addition, susceptibility to enzyme degradation was analyzed by means of limited proteolysis with thermolysin. Mutants which retained strong inhibitory activity were cleaved by thermolysin only at the reactive site, in the same way as native SMPI. The mutants which showed weak inhibitory activity underwent rapid degradation. These results were consistent with the substrate specificity of thermolysin. Based on these results, the reactive site of SMPI was identified as Cys64-Val65.

Mutational analysis of the reactive site loop of Streptomyces metalloproteinase inhibitor, SMPI.

Streptomyces metalloproteinase inhibitor (SMPI) is the only inhibitor to show "standard mechanism inhibition" against metalloproteinases. SMPI is a globular protein with an exposed loop containing the reactive site, C64-V65. To analyze the importance of basic residues in the reactive site loop of SMPI, mutants were constructed for R60, K61, and R66 (R60A, K61A, R66A, R60/K61A, 60/61/66A, and 60/61/66E). The mutants involving only R60, K61, and R60/K61 residues, respectively, showed strong inhibitory activity and were stable against enzyme activity. Both the triple mutants showed very weak inhibitory activity and underwent rapid degradation. The addition of basic residues to the loop (V62R and T63R) did not cause any further increase in inhibitory activity. These results suggest that basic residues in the reactive site loop play some role in maintaining a stable enzyme-inhibitor complex. The R66 mutant showed reduced activity and was rapidly degraded by enzymes. It was concluded that R66 is essential for maintaining a strong hydrophobic interaction with the S1' hydrophobic pocket of the enzyme. To investigate the roles of the disulfide bridge and the P68 residue near the reactive site, C64/69S and P68T mutants were constructed. These mutants showed very weak inhibitory activity and were rapidly degraded by enzymes. These results suggest that the disulfide bridge and P68 residue are very essential for SMPI to function as an inhibitor.