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Biomedical subjects

T Murachi

Publications and source records attributed to T Murachi.

At least 55 records · Page 3Linked to original sources
PubMed1989

Intracellular regulatory system involving calpain and calpastatin.

Seven years have elapsed since the terms calpain and calpastatin were introduced. During these years, significant progress in research has been recorded. Thus, cloning and sequencing of cDNAs for calpains I and II and calpastatin have established amino acid sequences of these molecules. Structure-function relationship of calpastatin has been studied using mutated cDNAs expressed in E. coli. Interleukin 2 receptor-linked expression of calpastatin in HTLV-I-infected T-cells has been reported. Evidence for Ca2+-induced translocation of calpain to the cell membrane, followed by its autolytic activation, has been discussed. A great varieties of proteins such as several kinases, membrane and cytoskeletal proteins, and hormone receptors have been reported to be susceptible to calpains. This paper is to summarize our current knowledge on chemistry and biology of calpain and calpastatin and thereby to speculate the true function of calpains and their regulatory mechanisms.

Amino Acid Sequence↗
PubMed1989

Multiple forms of calpastatin in pig brain.

Pig brain was found to contain two calpain-specific, heat-stable inhibitory fractions which could be separated by DEAE-cellulose chromatography. CS-0.1, which was eluted from the column at 0.1 M NaCl, was identified as an ordinary, well-known calpastatin. CS-0.2, eluted at 0.2 M NaCl, was different from CS-0.1 in that it inhibited calpain 1 more strongly than calpain II and that it did not cross-react with anti-calpastatin antibodies. Partial purification indicated that CS-0.2 contained inhibitor proteins smaller than ordinary calpastatin, but whether they are the products derived from CS-0.1 or entirely different genetic products has not yet been determined.

Animals↗
PubMed1989

Cloning and expression of the genes for calpains and calpastatins.

Calpain and calpastatin are known to be very widely distributed in animal cells. Although the physiological roles of calpain have not yet been clarified, the proteinase-proteinase inhibitor system has been suggested to play important roles in various cellular functions coupled with Ca2+ mobilization. By nucleotide sequencing of the cloned cDNAs, the primary structures of calpains and calpastatins have recently been disclosed. The expression of the genes for calpains and calpastatins in vitro as well as in vivo is being studied using cDNAs and their fragments. This paper reviews several topics on the recent progress of calpain research.

Amino Acid Sequence↗
PubMed1988

Analysis of structure-function relationship of pig calpastatin by expression of mutated cDNAs in Escherichia coli.

Structure-function relationships in pig calpastatin were investigated. Calpastatin is an endogenous inhibitor protein specifically acting on calpains (Ca2+-dependent cysteine endopeptidases). We recently cloned and sequenced the cDNA for pig heart calpastatin and determined the amino acid sequence of the molecule from the nucleotide sequence. Various deletion mutants in one of the four internally repetitive domains (Domain 3, approximately 140 amino acid residues) were created by in vitro site-directed mutagenesis of a cloned cDNA fragment and expressed in Escherichia coli. Deletion of a conserved region on either the amino-terminal or carboxyl-terminal side caused a drastic loss of inhibitory activity against calpain I (low Ca2+-requiring form) and, to a lesser degree, against calpain II (high Ca2+-requiring form). Inhibitory activities were below the detectable level in mutants deleted further toward the central region. Substitution of two amino acids in the latter region of the wild-type Domain 3 protein caused a drastic loss of activity against both calpains. The creation of lowered affinity inhibitors enabled us to perform a conventional kinetic analysis which showed the mode of inhibition to be competitive. Prediction of the secondary structure of Domain 3 suggests that both the amino- and carboxyl-terminal conserved regions form alpha-helical structures, which are largely located in the interior of the calpastatin molecule, whereas the central region does not form alpha-helix or beta-structure. The central region contains a 12-residue consensus sequence common to Domains 1, 2, and 4, and this portion is predicted to be located on the surface of the calpastatin molecule. These results suggest that the central conserved region of each domain of calpastatin is an area for direct interaction either with the active center of calpain or a region in close proximity, and the rest of the domain is a region stabilizing the functionally important tertiary structure of the domain.

Amino Acid Sequence↗
PubMed1988

Molecular and catalytic characterization of intact heterodimeric and derived monomeric calpains isolated under different conditions from pig polymorphonuclear leukocytes.

Evidence is presented of polymorphonuclear (PMN) cells derived from pig peripheral blood containing two molecular species of Ca2+-dependent cysteine endopeptidases, calpains I and II, which require low and high concentrations of Ca2+, respectively, for activation. Calpains I and II, purified from PMN homogenates, are heterodimers consisting of 83 plus 29 kDa and 80 plus 29 kDa subunits, respectively, which can be identified by using subunit-specific antibodies and which are identical with those of calpain species in other pig tissues and cells hitherto reported. However, a 70-kDa calpain can also be detected when pig PMN cells are disrupted by the nitrogen cavitation method under rather mild conditions, i.e., with minimal destruction of the lysosomes. Lines of evidence are presented showing that the 70-kDa species is devoid of the light subunit, that it is artificially derived from naturally occurring heterodimeric calpain I, and that the PMN cells before disruption contained no such monomeric form. The isolated 70-kDa calpain I, or monomeric artifact, requires only 1 microM Ca2+ for half-maximal activation, and it is less pH stable and much less heat stable than the parent heterodimeric calpain I. A possible mechanism for the production of this artifact is discussed.

Amino Acids↗
PubMed1988

Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis.

Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed. The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues. A unique N-terminal domain is followed by four mutually homologous domains. The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0%. The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues. The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences. It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.

Amino Acid Sequence↗
PubMed1988

Fucosylated type-2 chain polylactosamine antigens in human lung cancer.

Four specific monoclonal antibodies (MAbs) have been used to study distributions of fucosylated type-2 chain polylactosamine antigens, Lex, poly Lex, Ley and sialylated Lex-i antigens, in human lung cancer tissues and in the serum of patients with lung cancers. Radioimmunoassay frequently showed abnormally high antigen levels in the sera of 66 lung cancer patients tested. When histological typing was performed, high serum levels of the above 4 antigens were most frequently observed in patients with adenocarcinoma of the lung; i.e., after combining the results from the 4 antigens, 75% of the sera from patients with lung adenocarcinoma were positive (50% in the case of large-cell carcinoma, 30% in the case of squamous-cell carcinoma and 27% for small-cell carcinoma). Among the 4 antigens, the sialylated Lex-i antigen had the highest positive incidence, 58%, in the sera of patients with adenocarcinoma of the lung, compared to 33% for Ley, 29% for poly Lex, and 8% for Lex antigen. On the other hand, when the distributions of these antigens in the lung cancer tissues of 42 patients were studied by immunohistological techniques, the Ley antigen had the highest positive incidence, 100%, in lung adenocarcinoma tissues, poly Lex antigen had 86%, sialylated Lex-i antigen had 71%, and Lex antigen had 29%. In cancer tissues, the incidence of non-sialylated antigens, such as Ley, poly Lex and Lex antigens, often exceeds the positive incidence of the sialylated antigen, but the sialylated form of the antigen, such as sialylated Lex-i antigen, appears more often than the non-sialylated form in patients' sera.

Adenocarcinoma↗
PubMed1988

Glycolipid-directed FH6 monoclonal antibody recognizes high molecular weight glycoprotein antigen carrying sialyl Lex-i determinant in the culture supernatant of PC-9 cells.

The properties of the antigen recognized by monoclonal antibody FH6 have been analyzed. FH6 was originally generated against a glycolipid, i.e. a difucoganglioside isolated from human colonic adenocarcinoma, and specifically reacts with sialyl Lex-i determinant. Several culture supernatants of human carcinoma cell line cells were found to have high levels of FH6-reactive antigen, and PC-9, a human lung carcinoma cell line was used for the analysis. A solid-phase sandwich radioimmunoassay was performed to detect the antigen. The antigenic activity was extractable in 0.6 M PCA or 7% TCA, and was sensitive to mild alkaline treatment and to Pronase digestion. Most of the antigen was eluted in the void volume of a Sepharose CL-2B column, which indicates that its molecular weight is greater than several million. It was eluted from a DEAE-cellulose column at a NaCl concentration in the range of 0.2-0.25 M. The immunoaffinity-purified antigen has a high carbohydrate content of more than 80%. These data indicate that the antigen recognized by FH6 in the culture supernatant of PC-9 is not a glycolipid, but a high molecular weight glycoprotein which could be referred to as a mucin, or a proteoglycan, which contains keratan-sulfate like glycosaminoglycan chains, as judged from the results of the glycosidase treatments.

Antibodies, Monoclonal↗
PubMed1988

Calpain activates two transglutaminases from porcine skin.

Two transglutaminases (TGase) with estimated molecular weight of 55,000 (55-K TGase) and 120,000 (120-K TGase) were partially purified from the cytosolic fraction of porcine skin (epidermis-rich preparation) using DEAE-cellulose and gel-filtration chromatographies. The enzyme activities of both transglutaminases were enhanced more than 20-fold by treatment with calpain (Ca2+-dependent cysteine proteinase) in the presence of Ca2+, and this enhancement was inhibited by adding EDTA, leupeptin, or an endogenous calpain-specific inhibitor protein (calpastatin). 55-K TGase was effectively activated by a smaller amount of calpain than was 120-K TGAse, while known activating reagents such as thrombin and dimethyl sulfoxide or heat treatment preferentially activated 120-K TGase. One of the physiological functions of calpain in the epidermis may be the activation of epidermal transglutaminases.

Animals↗
PubMed1988

Creatine kinase-inhibiting monoclonal antibodies: preparation and characterization of porcine MM isoenzyme-specific antibodies.

Five monoclonal antibodies (CKM-B07, F12, D08, H09 and G01) against porcine creatine kinase (CK; EC 2.7.3.2) MM isoenzyme, which inhibit the enzymatic activity, were prepared. The hybridomas which produced monoclonal antibodies were screened by direct measurement of the inhibitory activity of their culture supernatant. Only two of them, however, were found to be measurable by an enzyme-linked immunosorbent assay with porcine CK-MM as an antigen. CKM-G01 inhibited 100% porcine CK-MM activity, while the others, 73-87%. On the other hand, only CKM-H09 inhibited porcine CK-BB activity (15%). CKM-F12 and D08 inhibited more than 50% CK-MB activity, whereas they did not inhibit CK-BB activity. The monoclonal antibodies were also tested for bovine, rabbit and human CK-MM. All the antibodies inhibited bovine and human CK-MM activity as well. In particular, CKM-G01 was found to exhibit more than 98% inhibition of all CK-MM activity tested, indicating that a common or very similar epitope which affects the activity is present on these enzymes. Admixing of CKM-B07 with other antibodies effected synergisms in inhibition, not only to porcine CK-MM activity but also to human CK-MM activity. A mixture of CK-B07 and G01 inhibited 100% human CK-MM activity, suggesting applicability of these monoclonal antibodies to clinical laboratory diagnosis.

Animals↗
PubMed1988

Identification of both calpains I and II in nucleated chicken erythrocytes.

Chicken erythrocytes were found to contain two species of calpains which differ in elution profile from DEAE-cellulose and in Ca2+ requirement. After partial purification, one of them was half-maximally activated by 10 microM Ca2+ and the other by 180 microM Ca2+. The low- and high-Ca2+-requiring proteases cross-reacted only with the respective monospecific antibodies for mammalian calpain I and calpain II, respectively. Approximately 5 times more calpain I than calpain II is present in chicken erythrocytes. By immunoelectrophoretic blot analysis, both calpains I and II from chicken erythrocytes were proved to be heterodimers composed of 76 and 28 kDa, and 80 and 28 kDa subunits, respectively. Our present finding that the heavy subunit of calpain I is smaller than that of calpain II is noteworthy, since the opposite is known to be true of various mammalian calpains. An immunological study has revealed that the calpain I newly found in chicken erythrocytes is not derived from calpain II. Thus, the co-existence of calpains I and II in one animal species also holds in chickens, contrary to the previously advocated notion that chickens have only one type of calpain.

Animals↗
PubMed1988

Purification and characterization of calpains from pig epidermis and their action on epidermal keratin.

Two forms of Ca++-dependent cysteine proteinases, calpain I, requiring low Ca++ (microM concentration), and calpain II, requiring high Ca++ (mM concentration), were purified from the cytosolic fraction of pig epidermis. Calpains I and II were separated on DEAE-cellulose chromatography, and thereafter they were purified by separate but almost identical procedures, which included chromatographies on Sephacryl S-300, Blue Sepharose CL-6B, and DEAE Bio-Gel A. Purified calpains I and II required 10 and 450 microM Ca++ for half-maximal activation, respectively, and had an optimal pH of 7.0 to 8.0. Both enzymes were heterodimers and composed of one heavy subunit (83 kDa for calpain I and 80 kDa for calpain II) and one light subunit (29 kDa for both enzymes). The action of calpains I and II on keratin extracted from the same tissue was studied. Both enzymes rapidly cleaved keratin into small fragments. The cleavage depends on Ca++ and could be blocked by leupeptin and calpastation, an endogenous calpain-specific inhibitor, which was also found in the cytosolic fraction of pig epidermis and partially purified.

Animals↗
PubMed1988

Distribution and expression of calpastatin in human hematopoietic system cells.

Thirteen cell lines of the human hematopoietic system were tested for expression of calpastatin at three different levels (mRNA, product and activity). The amount of calpastatin product and its inhibitory activity varied markedly depending upon cell types or human T-cell leukemia virus type I infection, although there were difference in the expression at the transcriptional level. Cell lines with high content of calpastatin comprised T-cell lineage infected by human T-cell leukemia virus type I, B-cell line and myelocytic leukemia cell lines. Non-infected T-cell lines and null cell line revealed themselves as poor in both calpastatin products and inhibitor activities. Two different molecular forms of calpastatin were observed: a 102-kDa form in T- and B-cell lines and a 94-kDa form in one of myelocytic cells. Three mRNA species of calpastatin (3.8, 3.0 and 2.5 kb) were identified in human hematopoietic system cells.

Calcium-Binding Proteins↗
PubMed1988

Distribution of calpains and calpastatin in human blood cells.

The occurrence and molecular sizes of calpains and calpastatin in the lysates of human erythrocytes, platelets, lymphocytes/monocytes, and polymorphonuclear cells were studied by immunoelectrophoretic blot analysis. The basic uniformity among these cells of the 85-kDa and 83-kDa heavy subunits of low- and high-Ca2+-requiring calpains I and II, respectively, and of the 29-kDa light subunit was confirmed. Molecular diversity of calpastatin species, ranging from 70 kDa to 107 kDa, among different blood cells was also shown. The obtained data are consistent with those known for other animal tissues, thus settling hitherto uncertain or rather controversial issues on the distribution of calpains and calpastatin in human blood cells.

Blood Platelets↗
PubMed1988

Evidence that calpains and elastase do not produce the von Willebrand factor fragments present in normal plasma and IIA von Willebrand disease.

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Because calpains and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze von Willebrand factor (vWF) in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. Using monoclonal epitope mapping, we have examined the proteolysis of the vWF subunit by porcine calcium-activated neutral proteases (calpains) and human leukocyte elastase to determine whether they produce the vWF proteolytic cleavage products seen in normal individuals and IIA vWD. Purified vWF was digested with porcine calpains I and II. We found no difference in the size, location, and quantity of the fragments produced by calpain I v calpain II. New fragments were detected of approximately 200, 170, 150, and 125 Kd. There was no evidence for generation of the native 140 and 176 Kd fragments. Some loss of the native fragments was seen, which suggests that they were further cleaved. Epitope mapping of the 170- and 150-Kd calpain-cleaved fragments revealed them to be from different parts of the molecule than the regions from which the native 176- and 140-Kd fragments derived. This was further supported by determination of the amino-terminal sequence of the calpain-cleaved 170- and 150-Kd fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 170/165, 145/140, and 130/125 Kd. No generation of native fragments was detected. Monoclonal epitope mapping of the 145/140-Kd elastase-cleaved band proved that it derived from the carboxyl-terminal portion of the vWF molecule, whereas the native 140-Kd fragment is derived from the amino-terminal end. Neither calpains nor human leukocyte elastase produced the proteolyzed fragments present in normal and IIA vWD and, therefore, probably do not cause the loss of large multimers that is seen in that disorder.

Amino Acid Sequence↗