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Biomedical subjects

T Murachi

Publications and source records attributed to T Murachi.

At least 127 records · Page 7Linked to original sources
PubMed1984

Highly sensitive flow injection analysis of glucose and uric acid in serum using an immobilized enzyme column and chemiluminescence.

A method for the flow injection analysis of glucose and uric acid in serum using immobilized enzymes in column form and chemiluminescence detection is described. The method is based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide which is produced by the action of the respective oxidases on glucose and uric acid. Glucose or uric acid in serum were determined with 1 microliter of the sample at a speed of 120 samples/h without carryover and at an assay time of approximately 10 s. The immobilized glucose oxidase column measured only 1.0 X 5 mm, and the immobilized uricase column 1.0 X 20 mm. The present method gave perfect linearity of the data up to 4.0 g glucose per liter or 0.10 g uric acid per liter with satisfactory precision, reproducibility, and accurate reaction recoveries. Furthermore, the present method was hardly affected by ascorbic acid, while the peroxidase-linked colorimetric method is usually influenced significantly by ascorbic acid. Both column reactors showed good operational stability for a 2-month period, during which time they were repeatedly used for analyses over 2000 times. The results on glucose and uric acid correlated satisfactorily with those obtained by other well-established methods.

Blood Glucose↗
PubMed1983

Automated assay of creatinine in serum as simplified by the use of immobilized enzymes, creatinine deiminase, and glutamate dehydrogenase.

A method for the automated analysis of creatinine in serum using immobilized enzymes in column form which does not require a blank correction and can be run in a single buffer of pH 7.5 is described. The method was based on the determination of ammonia formed by the action of creatinine deiminase. Endogenous ammonia in serum was removed by an immobilized glutamate dehydrogenase column prior to the action of creatinine deiminase also immobilized and used in column form. The present method was found to give perfect linearity of the data up to 0.10 g creatinine/liter with satisfactory precision, reproducibility, and accurate reaction recoveries. The immobilized enzyme reactor unit showed good operational stability for a 2-month period, during which time it was repeatedly used for analyses over 2000 times. The results correlated satisfactorily with those obtained by other well-established methods.

Adult↗
PubMed1983

The appearance of a 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) in rat liver after the administration of phenylhydrazine.

A 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) was found in the cytosol of anemic rat liver. When phenylhydrazine hydrochloride was continuously administered to rats, a 280,000-dalton calpain inhibitor that existed originally in the liver gradually disappeared within two weeks and, concomitantly, a 34,000-dalton inhibitor appeared. The purified 34,000-dalton inhibitor resembles 280,000-dalton inhibitor in that both are heat-stable proteins and do not inhibit papain and trypsin. Unlike the protomers of a 280,000-dalton inhibitor, 34,000-dalton inhibitor does not show any sign of self-association.

Anemia↗
PubMed1983

Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney.

Two molecular species of calpain (Ca2+-dependent cysteine proteinases) were concurrently purified from rat kidney, both to homogeneity. Calpain I and calpain II having low and high Ca2+ requirements, respectively, were clearly separated on DEAE-cellulose chromatography at pH 7.5, and thereafter they were purified by separate but almost identical procedures which included (NH4)2SO4 fractionation and successive chromatographies on TSK-Gel G 3000 SWG, blue Sepharose CL-6B, and DEAE-Bio-Gel A. The purification folds and activity yields were 6170-fold and 17.8% for calpain I and 4160-fold and 11.9% for calpain II. Ca2+ concentrations for half-maximal activation were 2 microM for calpain I and 200 microM for calpain II. The specific activity of calpain II on casein as the substrate was more than twice higher than that of calpain I. Both enzymes are heterodimers, each composed of 80,000-Da and 25,000-Da subunits. The amino acid compositions of calpain I and calpain II are very similar but not identical. Calpain II is more acidic (pI 4.6) than calpain I (pI 5.3). This paper is the first to describe parallel isolation and characterization of low and high Ca2+-requiring proteases from one single nonmuscular tissue.

Amino Acids↗
PubMed1983

Structural requirements for hemolytic activity of F-glycoprotein of HVJ (Sendai virus) studied by proteolytic dissection.

The structure-function relationship of F and HN glycoproteins of HVJ were studied by proteolytic dissection. Three types of effects on the biological activity and structure of the virus particles were observed. First type of effect is preferential inactivation of biological activities related to F glycoprotein, such as hemolytic and cell fusion-inducing activities. Among enzymes which exert such effects, trypsin split F1 subunit to F1a (32,000 daltons) and F1b (19,000 daltons). By N-terminal determination, F1a was found to be derived from the N-terminal segment of F1, whereas F1b seems to correspond with the C-terminal segment of F1. Chymotrypsin and thermolysin digestion resulted in decreases in molecular weight of F1 subunit by about 3,500 daltons and 2,500 daltons, respectively. This splitting was found to occur near the N-terminus of F1, since new N-terminal amino acids were identified from the modified F1's. The second type of effect is characterized by specific splitting (for example, by a Staphylococcal proteases) of HN glycoprotein without affecting F protein. The third type has no apparent effect on the biological activities of the virion, although slight structural change of F glycoprotein was noted in some case. Exposure of the N-terminal segment of F1 to the surrounding aqueous medium despite its highly hydrophobic nature is shown by its easy splitting by aminopeptidase M, chymotrypsin and thermolysin. Based on these and previously published results, we hypothesize direct interaction of the hydrophobic segment with the lipid bilayers of the target cell membrane as an important step in fusion reactions between the viral envelope and plasma membranes.

Hemolysis↗
PubMed1983

Similarity and dissimilarity in subunit structures of calpains I and II from various sources as demonstrated by immunological cross-reactivity.

The structural relationship between calpain I (low Ca2+-requiring) and calpain II (high Ca2+-requiring) molecules and their respective larger (80K) and smaller (30K) subunit proteins of several non-muscular tissues and cells was studied by testing immunological cross-reactivities. In addition to qualitative analyses by a conventional double immunodiffusion method, quantitative data were obtained, for the first time, by enzyme-linked immunosorbent assays using affinity-purified anticalpain I and anti-calpain II immunoglobulins. The enzyme sources included rat kidney, porcine kidney and erythrocytes, and human erythrocytes. It was concluded that the 30K subunits are immunologically almost indistinguishable between calpains, either I or II, not only from the same but also from different sources, while the 80K subunits of different origins are immunologically related to variable extents but always with discrimination between calpain I and calpain II. Similarity of the 30K subunit proteins and dissimilarity of the 80K counterparts were further substantiated by their chromatographic and electrophoretic behaviors.

Animals↗
PubMed1983

Suppressor cell induction factor: a new mediator released by stimulated human lymphocytes and distinct from previously described lymphokines.

Suppressor cell induction factor (SIF) was produced by alloantigen-stimulated human peripheral blood lymphocytes, and it activated human T cells to become effective suppressors of the mixed lymphocyte reaction (MLR). The activity of SIF was resistant to 56 degrees C and to pH 2, and was precipitated by 50 to 80% saturated ammonium sulfate. SIF had a m.w. range, as determined by gel filtration, of 18,000 to 29,000; it did not bind to DEAE cellulose columns; and it was recovered in the pH range from 6.9 to 7.3 on isoelectric focusing. SIF was biochemically separable from IL 2, BF, IFN-gamma, and CSF. Furthermore, IL 2 activity was completely removed by absorption of MLC supernatants by murine cytotoxic T lymphocyte line (CTLL) cells, whereas SIF activity was unabsorbable, thus distinguishing SIF from IL 2. In addition, antiviral activity of MLC supernatants was completely abolished by anti-human IFN-gamma serum, whereas SIF activity was unaffected by this antiserum, thus distinguishing SIF from IFN-gamma. Since treatment of these supernatants with antiserum against human lymphoblastoid cell IFN(alpha/beta) had no effect on either antiviral or SIF activities in these supernatants, SIF was also distinguishable from IFN alpha/beta. These results indicate that SIF is a distinct new lymphokine with the ability to induce suppressor function in human T cells.

Colony-Stimulating Factors↗
PubMed1983

A new mediator (suppressor cell induction factor) activating T cell-mediated suppression: characterization of suppressor cells, kinetics of their generation, and mechanism of their action.

Human T cells precultured in MLC supernatant (SIF) could suppress responder cells in MLC. SIF-activated cells expressed suppressor effects equally on both autologous and allogeneic responder cells in MLC. The suppression observed was not due to cell crowding, cell death, depletion of nutrients, absorption of IL 2, alternation in the kinetics of MLC response, production of soluble inhibitory factor, or generation of cytotoxic cells. In this system, suppressor cells were generated after 5 days of culture, and Ia-positive cells were required in the induction of suppressor cells. Suppressor cells themselves, however, were characterized as Ia-negative subsets of T cells; they could be derived from both Fc-IgG receptor-positive and -negative subsets of T cells. Suppressor activity of SIF-treated cells on the MLC response was sensitive to mitomycin C treatment. They apparently exerted their suppressor effect on some early phase of the MLC response. This early event was almost complete within 48 hr after the initiation of MLC. The possibility that SIF may be involved in T-T interactions required in the generation of effective suppression is discussed.

Animals↗
PubMed1982

Effect of bongkrekic acid, a product of Pseudomonas cocovenenans, on thiol proteases.

The in vitro effect of bongkrekic acid on stem bromelain, papain and ficin was studied. The hydrolysis of casein by these enzymes was inhibited by bongkrekic acid, but the inhibition was always incomplete even with a large excess of the effector. Using a fully activated specimen of stem bromelain, purified on an organomercurial agarose affinity column, the inhibition by bongkrekic acid was not stoichiometric. The SH group of cysteine remained intact after incubation with an excess of bongkrekic acid at 24 degrees C for 20 min. However, partial inhibition of stem bromelain by bongkrekic acid was reversed by incubation at 37 degrees C for 5 min with 5mM cysteine or 2-mercaptoethanol. Ethylene glycol and glycerol had no such restorative effect. These results indicate that molecules of bongkrekic acid are non-covalently bound to a thiol protease, only partially and reversibly shielding its essential SH group.

Anti-Bacterial Agents↗
PubMed1982

Purification and some properties of human erythrocyte calpastatin.

Calpastatin, an endogenous inhibitor protein specifically acting on calpain [EC 3.4.22.17; Ca2+-dependent cysteine proteinase], was purified to apparent homogeneity from the cytosol fraction of human erythrocytes. The yield was 0.38 mg from 400 ml of blood. The purification procedures included DEAE-cellulose and Ultrogel AcA34 gel chromatographies followed by heat treatment and a final gel chromatography on Sephacryl S-200, from which calpastatin was eluted at a position corresponding to a 280,000-dalton molecular mass. The heat treatment at 100 C for 15 min at pH 7.5 very effectively removed contaminant proteins. The homogeneity of the final product was demonstrated on polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, giving a single protein band with a calculated molecular weight of 70,000. After re-extraction from the gel, the 70,000-dalton protein readily reassociated to give a 280,000-dalton peak upon Sephacryl S-200 chromatography with a recovery of total inhibitory activity of 42%. Purified calpastatin had an isoelectric point at pH 4.55. It had glutamine as the amino-terminal residue. Amino acid analyses revealed that it contained relatively large amounts of proline, glutamic acid, and lysine, smaller amounts of aromatic amino acids, notably no tryptophan, and no amino sugars. The content of half-cystine was less than one per 643 amino acid residues. These features are in general agreement with those of the reported amino acid composition of Ca2+-protease inhibitor from chicken skeletal muscle [Ishiura et al. (1982) Biochim. Biophys. Acta 701, 216-223], although these inhibitors were found to be definitely different in several other respects. Human erythrocyte calpastatin could inhibit not only calpain of the same origin but also calpains having low and high Ca2+-sensitivity from rat liver.

Amino Acids↗
PubMed1981

The in vitro and in vivo behavior of urokinase immobilized onto collagen-synthetic polymer composite material.

With a view to developing biomaterials for semipermanent substitution, we have studied a composite material constituted with collagen and a synthetic polymer which possesses high tissue compatibility. This collagen-synthetic polymer composite was applied as a support for immobilization of enzymes for the purpose of providing a material surface with biological function. The enzymes, urokinase and trypsin, were successfully bound to the collagen membrane layer which had been activated by acyl azide formation of its carboxyl groups. The enzyme-bearing composite material showed excellent catalytic activity toward a protein substrate as well as a low-molecular-weight synthetic substrate. The immobilized urokinase was characterized enzymatically and compared with native urokinase. The apparent affinity of immobilized urokinase for the substrate was slightly decreased, but its intrinsic kinetic properties were not significantly affected. No decrease in its esterase activity was observed both on repeated use and on long-term storage, and its fibrinolytic activity was stable on heat or disinfection treatment. When this urokinase-bearing composite material was applied into rabbit blood vessels, its in vivo fibrinolytic activity was maintained. Thus, enzyme-collagen-synthetic polymer composites may find wide application for biomaterials and artificial organs as functional biomaterials.

Animals↗
PubMed1981

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin).

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.

Calcium↗