Search PubMed⌕ Search

Biomedical subjects

T Murachi

Publications and source records attributed to T Murachi.

At least 19 recordsLinked to original sources
PubMed1992

Expression of calpain II gene in human hematopoietic system cells infected with human T-cell leukemia virus type I.

We examined the distribution of calpains I and II in human hematopoietic system cell lines by Western and Northern blot analyses and enzyme activity assay. Expression of calpain I, a low Ca(2+)-requiring cysteine protease, was observed in all human T-cell lines tested. By contrast, expression of calpain II, a high Ca(2+)-requiring form, in human T-cells was closely correlated with human T-cell leukemia virus type I (HTLV-I) infection, which is known to result in the expression of adult T-cell leukemia-associated antigens, interleukin-2 (IL-2) receptor alpha, and Ca(2+)-dependent cell proliferation. Specific expression of calpain II in HTLV-I-infected cells occurred at the mRNA level. Furthermore, expression of calpain II in human natural killer-like cells was augmented by HTLV-I pX gene transfection. In HTLV-I-infected cells, the trans-acting transcriptional activation of the long terminal repeat and control elements for the IL-2 receptor alpha, c-fos, and granulocyte-macrophage colony-stimulating factor genes by the Tax from the pX gene is already known. Our results suggest that the similar trans-activation occurs to the calpain II gene in HTLV-I-infected hematopoietic system cells.

Blotting, Western↗
PubMed1992

Characterization of proteoglycan degradation by calpain.

Degradation of cartilage proteoglycans was investigated under neutral conditions (pH 7.5) by using pig kidney calpain II (EC 3.4.22.17; Ca(2+)-dependent cysteine proteinase). Aggregate and monomer degradation reached a maximum in 5 min at 30 degrees C when the substrate/enzyme ratio was less than 1000:1. The mode of degradation was limited proteolysis of the core protein; the size of the products was larger than that of papain-digested products and comparable with that of trypsin-digested products. The hyaluronic acid-binding region was lost from the major glycosaminoglycan-bearing region after incubation with calpain II. Calpains thus may affect the form of proteoglycans in connective tissue. Ca(2+)-dependent proteoglycan degradation was unique in that proteoglycans adsorb large amounts of Ca2+ ions rapidly before activation of calpain II: 1 mg of pig cartilage proteoglycan monomer adsorbed 1.3-1.6 mu equiv. of Ca2+ ions before activation of calpain II, which corresponds to half the sum of anion groups in glycosaminoglycan side chains. This adsorption of Ca2+ was lost after solvolysis of proteoglycan monomer with methanol/50 mM-HCl, which was used to desulphate glycosaminoglycans. Therefore cartilage proteoglycans are not merely the substrates of proteolysis, but they may regulate the activation of Ca(2+)-dependent enzymes including calpains through tight chelation of Ca2+ ions between glycosaminoglycan side chains.

Animals↗
PubMed1992

Use of various types of column reactors for flow-injection analysis.

Two or three different kinds of immobilized enzymes can be aligned in a minireactor so that sequential enzymatic reactions are carried out from upstream to downstream during flow-injection analysis. A lactate oxidase-catalase reactor, used as precolumn for removing pre-existing lactate in serum before the lactose dehydrogenase (LDH) reactions, was useful for the determination of serum LDH activity, which did not require any blank correction. A sequential glutamate dehydrogenase-glutamate oxidase reactor was also useful for a novel chemiluminometric determination of ammonia. On the other hand, a co-immobilized creatininase-creatinase-sarcosine oxidase reactor, in spite of containing creatininase which catalyses the reversible reaction, was the most efficient for the determination of serum creatinine.

Ammonia↗
PubMed1991

Structure of the active 27-residue fragment of human calpastatin.

A synthetic 27-residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well-conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO-d6 by two-dimensional 1H NMR spectroscopy. Although regular secondary structures such as alpha-helix and beta-sheet were not found, the region from Ile18 to Arg23 formed a well-defined structure with a type I beta-turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.

Amino Acid Sequence↗
PubMed1991

Phosphorylation and subcellular distribution of calpastatin in human hematopoietic system cells.

Calpastatin is an endogenous inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2(+)-dependent cysteine proteinase). The phosphorylation of calpastatin was investigated in human hematopoietic system cell lines. Microheterogeneity of calpastatin was observed, in which 118- and 116-kDa forms were named calpastatin a and b, respectively. The phosphorylation of both calpastatins was identified in all cell lines examined and occurred mainly at serine residues with trace amounts of phosphothreonine in vivo. The incubation of cells with 12-O-tetradecanoylphorbol-13-acetate increased the incorporation of 32P-orthophosphate into calpastatin a. Two-dimensional maps of 32P-labeled phosphopeptide from both calpastatins were identical except for additional minor spots for calpastatin a. [35S]methionine-labeled calpastatins a and b were localized mainly in the cytosol, and only 6% of cellular calpastatins were detected in the membrane fraction. By contrast, more than 30% of the 32P-labeled calpastatins a and b were distributed in the membrane fraction. Thus, the phosphorylation of calpastatin may be involved in regulating the calpain-calpastatin protein kinase system by its subcellular distribution.

Amino Acid Sequence↗
PubMed1991

Determinations of lactate and lactate dehydrogenase activity in serum with the flow injection analysis system involving immobilized enzyme column and chemiluminescence.

The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods.

Autoanalysis↗
PubMed1991

Immunohistochemical and biochemical demonstration of calcium-dependent cysteine proteinase (calpain) in calcifying cartilage of rats.

Calpain is a Ca2(+)-dependent cysteine proteinase that has neutral pH optima. There are two classes of calpains that differ in their optimal calcium ion concentration for enzymatic activity. Calpain I requires a low concentration of Ca2+ for activation, and calpain II requires a much higher Ca2+ concentration. This report describes the immunohistochemical and biochemical demonstration of calpain II in calcifying cartilage in rats and also the degradation of the cartilage proteoglycan subunit by calpain II. Immunoperoxidase (peroxidase-antiperoxidase) staining of the frozen sections of the knee joint from 3-day-old and 6-day-old Wistar rats, using polyclonal antibodies against the respective heavy subunits of calpains I and II, showed positive staining only with the anti-calpain II antibody in the hypertrophic chondrocytes and surrounding cartilaginous matrix of the growth cartilage. Diethylaminoethyl-cellulose chromatography of the cartilaginous extract from 3-day-old rats showed a peak of caseinolytic activity attributable to calpain as well as an inhibitory peak of calpastatin, a specific inhibitor protein of calpains. Immunoblotting using the anti-calpain II antibody of the calpain peak demonstrated identity with the heavy subunit of calpain II (80 kDa). Proteoglycan-degrading activity of calpain was assessed using porcine kidney calpain II and the porcine articular cartilage proteoglycan subunit. After incubation in the presence of Ca2+, degradation of proteoglycan was demonstrated by the change of the elution position on Sepharose-2B chromatography. It is possible that calpain functions as one of the proteoglycan-degrading proteolytic enzymes of growth cartilage. Intracellular localization of calpain in hypertrophic chondrocytes also suggests a role in the hypertrophic process of the chondrocyte in growth cartilage.

Animals↗
PubMed1991

Absence of erythrocyte arginase protein in Japanese patients with hyperargininemia.

In Japan, hyperargininemia has been reported in only 5 unrelated families and four patients are alive at present. In this study we examined arginase protein in erythrocytes of these Japanese patients using two analytical methods of immunoblotting and two-dimensional gel electrophoresis. Immunoblotting study with anti-E. coli-expressed human liver arginase rabbit IgG revealed lack of cross-reacting materials in the erythrocyte lysates from these patients. On two-dimensional gels, arginase protein was detected in any control subject, but it was completely absent in all the patients studied. These results suggest that either arginase protein in erythrocytes is not produced or it is structurally labile in these patients.

Adult↗
PubMed1991

Direct measurement of calpastatin subtypes by sandwich enzyme immunoassay using monoclonal antibodies.

Six stable hybridoma cell lines secreting monoclonal antibodies to human calpastatin were established. All monoclonal antibodies belong to the IgG1 subclass and recognized different epitopes on calpastatin. At least two groups were distinguished; the first group was specific for muscle-type (M-) calpastatin and the second group recognized not only M-calpastatin but also erythrocyte-type (E-) calpastatin. The inhibitory effect of all monoclonal antibodies on calpastatin activity was relatively low even at high concentrations of antibodies. Enzyme immunoassay systems were developed for direct determination of calpastatin subtypes in human cells requiring no other sample treatment than the disruption of the cells. The assay methods were, in principle, based on the sandwich enzyme immunoassay using epitope-specific monoclonal antibodies. The enzyme immunoassay system for M-calpastatin was specific for M-calpastatin and could not detect E-calpastatin. The enzyme immunoassay system for total calpastatin detected not only M-calpastatin but also E-calpastatin. The sensitivity of these assay systems was 10 pmol l-1 of calpastatins. Antigenicity of calpastatins was found to be unchanged in the presence of EDTA and haemoglobin. Good reproducibilities of within-and between-assay series and excellent recovery of exogenous calpastatins from cell lysates were observed. From these results, it seems that our newly developed subtype-specific enzyme immunoassay systems for calpastatins are useful in biochemical studies and clinical testing for determination of calpastatin subtypes.

Antibodies, Monoclonal↗
PubMed1991

Molecular diversity of erythrocyte calpastatin.

According to the difference of molecular masses estimated by SDS-polyacrylamide gel electrophoresis, calpastatins are classified into two types, i.e. muscle type (110 kDa) and erythrocyte type (70 kDa). Muscle type calpastatin contains four internally repetitive sequences (Domains 1-4) and one nonhomologous sequence on the amino-terminal side (Domain L), whereas erythrocyte type lacks Domains L and 1. By immuno-blot analysis, chicken erythrocytes, nucleated cells, were found to contain muscle type calpastatin. In avian erythrocytes, diminution of the calpastatin molecule as in mammalian erythrocytes was not observed.

Animals↗
PubMed1991

Amelioration of cataracts and proteolysis in cultured lenses by cysteine protease inhibitor E64.

Cataracts were produced in cultured rat lenses by either 10 microM calcium ionophore A23187, 25 microM sodium selenite, or 30 mM xylose. E64, an inhibitor of cysteine proteases, such as calpain (EC, 3.4.22.17), reduced severity of cataract and proteolysis of crystallins when included at a 500 microM concentration in the culture medium along with cataractogenic agents. Calpain II enzyme activity and the amount of calpain antigen were decreased in the cytosol of cataractous lens. However, E64 caused an increase in the amount of an 80-kD calpain subunit associated with the ethyleneglycol-bis-(beta-aminoethylether) tetraacetic acid/ethylenediaminetetraacetic acid-washed insoluble proteins when lenses were incubated with cataractous agents. These data indicate that E64 was at least partially effective in inhibiting lens calpain, and that activation of lens calpain may involve binding to the insoluble fraction. These results provide strong evidence for the activation of calpain in rodent cataracts and suggest testing inhibitors of calpain as anticataract drugs.

Animals↗
PubMed1990

Expression of human liver arginase in Escherichia coli. Purification and properties of the product.

Arginase is an enzyme that catalyses the hydrolysis of arginine to urea and ornithine. It is abundantly present in the liver of ureotelic animals (i.e. those whose excretion is characterized by the excretion of uric acid as the chief end-product of nitrogen metabolism), but its purification has hitherto not been simple, and the yield not high. Starting with a partially truncated cDNA for human liver arginase recently made available, we constructed an expression plasmid that had tandemly linked tac promotors placed upstream of a full-length cDNA. By selecting Escherichia coli strain KY1436 as the host micro-organism, we established an efficient system for the production of human liver arginase protein. Chromatographies on CM-Sephadex G-150, DEAE-cellulose and Sephadex G-150, followed by preparative agar-gel electrophoresis, yielded 10 mg of apparently homogeneous enzyme protein from 1 g (wet wt.) of E. coli cells. E. coli-expressed human liver arginase had chemical, immunological and most catalytic properties indistinguishable from those of purified human erythrocyte arginase. However, E. coli-expressed arginase was a monomer of Mr 35,000, whereas the purified erythrocyte arginase was trimer of Mr 105,000. They differed also in pH- and temperature-stabilities. Gel-filtration experiments with these two purified arginases under various conditions, as well as with unfractionated human liver and erythrocyte cytosol preparations, indicated that the native form of human arginase should be of Mr 35,000, and that the trimeric appearance of human erythrocyte arginase after purification was an artifact of the purification procedures. It was thus concluded that, in Nature, the liver and erythrocyte arginases are identical proteins.

Amino Acid Sequence↗
PubMed1990

Characterization of a functional domain of human calpastatin.

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Amino Acid Sequence↗
PubMed1990

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum.

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.

Amino Acid Oxidoreductases↗
PubMed1990

Calpain II in two in vivo models of sugar cataract.

Cataracts were produced in rat lenses by either feeding a diet containing 50% galactose or by inducing diabetic condition by intravenous injection of streptozotocin. Proteolysis of crystallins, protease activity of calpain II enzyme (EC 3.4.22.17), and presence of calpain molecule (antigen) were determined at four cataract stages--I, cortical vacuoles, II, vacuoles plus hazy cortex, III, nuclear cataract, and IV, mature cataracts. Calpain activity was normal or moderately elevated at early stages of galactose and diabetic cataracts. Later stages III and IV showed proteolysis of lens crystallins, increased proportion of insoluble proteins, loss of calpain enzyme activity and calpain molecule from the soluble fraction, and reduced amounts of calpain associated with insoluble pellet. In galactose cataract, the largest increase in lens calcium were found when proteolysis was present. These results provide evidence for calpain-induced proteolysis of lens crystallins in two in vivo models of sugar cataracts in rodents.

Animals↗
PubMed1990

A flow injection analysis system involving immobilized NADH oxidase in column form for clinical analysis.

A highly sensitive FIA system for chemiluminometric determination of reduced coenzyme, NADH, was developed, using immobilized NADH oxidase from Brevibacterium ammoniagenes. The enzyme catalyzed the oxidation of NADH generating hydrogen peroxide which emitted chemiluminescence when mixed with luminol and potassium ferricyanide. The immobilized enzyme reactor was a mini-column, measuring 1 or 2 mm in inner diameter and 20 mm in length, and the sample volume was only 1 microliter per assay, with a feeding speed of one sample per min and a lowest detection limit of 10 pmol NADH. A FIA system was also developed for the determination of magnesium in human serum, using an enzyme column reactor with simultaneously coimmobilized hexokinase, D-glucose-6-phosphate dehydrogenase, and NADH oxidase. The performance of the system was as satisfactory as a routine colorimetric assay, but with much higher sensitivity.

Colorimetry↗
PubMed1990

Significant amount of multicatalytic proteinase identified on membrane from human erythrocyte.

Multicatalytic proteinase (MCP) was solubilized from human erythrocyte membrane with 0.1% Triton X-100 and purified to homogeneity using a combination of DEAE-cellulose, hydroxylapatite, and Ultrogel AcA34 chromatographies. This membranous MCP had similar properties to MCP purified in parallel from the cytosol. Both MCPs had a molecular mass of 570 kDa, were composed of apparently nine subunits of 22-36 kDa and had trypsin- and chymotrypsin-like activities. These activities were latent and required heating for the induction. However, slight differences were observed in the effects of reagents (DFP, monoiodoacetic acid, Mg2+, and Ca2+) between membranous and cytosolic MCP. The amount of MCP identified on membranes was estimated to be three-quarters or one-half of that found in the cytosol based on its trypsin- or chymotrypsin-like activity, respectively.

Catalysis↗