[Application of genetic engineering to laboratory medicine: an outline].
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Clinical Laboratory Techniques↗
Biomedical subjects
T Murachi
Publications and source records attributed to T Murachi.
All four internally repetitive domains of pig calpastatin possess inhibitory activities against calpains I and II.
Complementary DNA portions coding for each domain (domain L and internally repetitive domains, domains 1-4, each composed of approximately 140 amino acid residues) of pig calpastatin were subcloned into E. coli plasmids to express the respective portions of the proteinase inhibitor gene in bacteria. Cell extracts of E. coli harboring recombinant plasmids were assayed for calpain inhibition. All four internally repetitive domains showed inhibitory activities, essentially similar to one another, against calpains I and II. No inhibition was observed in the case of the N-terminal non-homologous domain (domain L). These results support our previous conclusion that the repetitive region is a functional unit of the proteinase inhibitor.
Amino Acid Sequence↗
Interaction of human calpains I and II with high molecular weight and low molecular weight kininogens and their heavy chain: mechanism of interaction and the role of divalent cations.
Calpain I prepared from human erythrocytes was half-maximally and maximally activated at 23 and 35 microM calcium ion, and two preparations of calpain II from human liver and kidney were half-maximally activated at 340 and 220 microM calcium ion and maximally activated at 900 microM calcium ion, respectively. High molecular weight (HMW) and low molecular weight (LMW) kininogens isolated from human plasma and the heavy chain prepared from these proteins inhibited calpain I as well as calpain II. The molar ratios of calpains to HMW kininogen to give complete inhibition of calpains were 1.4 for calpain I and 2.0 for calpain II, and those of calpains to heavy chain were 0.40-0.66 for calpain I and 0.85 for calpain II. LMW kininogen did not completely inhibit the calpains even with an excess amount of kininogen. The apparent binding ratio of calpain to HMW kininogen estimated from the disc gel electrophoretic analysis, however, was found to be 2:1, whereas those of calpain to LMW kininogen and of calpain to heavy chain were found to be 1:1. Calpains and kininogens failed to form complexes in the absence of calcium ion. In the presence of calcium ion, however, they formed the complexes, which were dissociable by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The minimum concentrations of calcium ion required to induce complex formation between calpain I and kininogens and calpain II and kininogens were 70 and 100 microM, respectively. Some other divalent cations such as Mn2+, Sr2+, and Ba2+ were also able to induce the complex formation between calpains and kininogens.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium↗
Platelet factor XIII is activated by calpain.
The action of calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase) on platelet factor XIII has been studied. Calpain I activated platelet factor XIII up to 76% of the maximum level observed with thrombin. Activation was accompanied by the limited proteolysis of the a subunit of platelet factor XIII to produce a 76 kDa fragment which was comparable to the proteolytic product by thrombin. Activation of platelet factor XIII by calpain was inhibited by EDTA, leupeptin, and endogenous calpain-specific inhibitor calpastatin. These findings suggest that calpain is responsible for the intracellular activation of platelet factor XIII.
Blood Platelets↗
Repetitive region of calpastatin is a functional unit of the proteinase inhibitor.
A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E. coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria. The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase. The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability. These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor.
Amino Acid Sequence↗
High-performance liquid chromatographic assay of transglutaminase and its application to the purification of human erythrocyte transglutaminase and platelet factor XIII.
A high-performance liquid chromatographic method was developed for the assay of transglutaminase [EC 2.3.2.13] activity. Casein and dansylcadaverine were used as substrates and the reaction was stopped by adding an excess amount of EGTA. Casein-bound dansylcadaverine was separated from free dansylcadaverine by high-performance liquid chromatography on a TSK SW gel column on the basis of the differences in the molecular weight and hydrophobicity. The sensitivity was approximately 0.04 nmol of casein-bound dansylcadaverine in the assay mixture. With this assay method, human erythrocyte transglutaminase and platelet factor XIII were purified by successive chromatographies on DEAE-cellulose and Sephacryl S-300, which were common for both enzymes, followed by Blue Sepharose CL-6B and DEAE Bio-Gel A for erythrocyte transglutaminase or Phenyl-Sepharose CL-4B for platelet factor XIII. The purification factors and activity yields were 15,300-fold and 22% for erythrocyte transglutaminase and 43.8-fold and 33% for platelet factor XIII.
Blood Platelets↗
Use of a bioreactor consisting of sequentially aligned L-glutamate dehydrogenase and L-glutamate oxidase for the determination of ammonia by chemiluminescence.
A chemiluminometric method for the automated flow injection analysis of ammonia is described. The essence of the invention is the use of a bioreactor consisting of both immobilized L-glutamate dehydrogenase (GLDH) and L-glutamate oxidase (GLXD), which are sequentially aligned in this order in a minicolumn measuring 2.0 X 20 mm. The unidirectional constant flow of liquid through the column reactor minimizes the reversed diffusion of the solutes so that the following sequence of reactions is ensured. Thus, ammonia to be determined is first transformed by GLDH into L-glutamate, which then produces hydrogen peroxide by GLXD. Hydrogen peroxide in the effluent from the column is then determined by its chemiluminescence upon admixing with luminol and potassium ferricyanide. The present method gives linearity of the standard curve for ammonia up to 1.0 mM. It is at least 100 times more sensitive than the conventional method for ammonia assay using ultraviolet absorption measurement.
Amino Acid Oxidoreductases↗
Specific localization of tissue-type transglutaminase in adrenocorticotropin-producing cells of the human pituitary gland as demonstrated by immunohistochemistry.
The distribution of transglutaminases, the Ca2+-dependent protein cross-linking enzymes, in the human pituitary gland was investigated by immunohistological methods using specific antibodies. Tissue-type transglutaminase was specifically localized in ACTH-producing cells, and the cells producing GH, PRL, TSH, FSH, and LH contained no appreciable amount of the enzyme. No detectable plasma-type transglutaminase (coagulation factor XIII) was found in pituitary tissue. In a previous study we demonstrated that ACTH-producing cells contain very little Ca2+-dependent proteinases (calpain), but a remarkable amount of their inhibitor, calpastatin. Pituitary gland cells producing hormones other than ACTH contained calpains, but no detectable calpastatin. These results collectively suggest that intracellular substrate proteins in ACTH-producing cells are protected from Ca2+-dependent degradation and are substrates for Ca2+-dependent cross-linking catalyzed by the tissue-type transglutaminase. In other pituitary gland cells, conversely, the intracellular substrate proteins are more likely to undergo Ca2+-dependent degradation than cross-linking.
Adolescent↗
Elucidation of calpain dependent phosphorylation of myosin light chain in human platelets.
Newly synthetized calpain inhibitors (CI-I approximately III) were used to prove potential participation of calpain in protein phosphorylation. CIs were about 1,000 times more potent against platelet calpain I than N-ethyl-maleimide (NEM) and an epoxy succinate derivative (E-64). CI-II inhibited 20K (myosin light chain) and 47K phosphorylation of Ca2+-stimulated lysed platelets as well as protein degradation (actin binding protein, P235). Both myosin light chain kinase (MLCK) and C-kinase dependent phosphorylation of 20K were inhibited by CI-II as demonstrated in phosphopeptides mapping. Electropermeabilized platelets (EP) were employed to examine the effects of CI-II on Ca2+ mediated reactions in non-lysed platelets. Phosphorylation of 20K and 47K induced by Ca2+ addition to EP was inhibited by CI-II, though secretory response was not modified. Only MLCK dependent phosphorylation of 20K was observed in Ca2+-activated EP, which was inhibited by CI-II. Collectively, the data indicated that calpain may activate both MLCK and C-kinase to phosphorylate 20K by partial proteolysis.
Acriflavine↗
[Physiological significance of the calpain-calpastatin system].
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Amino Acid Sequence↗
Monoclonal and polyclonal antibodies against porcine mitochondrial aspartate aminotransferase: their inhibition modes and application to enzyme immunoassay.
Monoclonal and polyclonal antibodies against porcine mitochondrial aspartate aminotransferase (m-AST) were prepared in order to study their effect on the kinetics of the enzyme and their possible use as diagnostic reagents. The most stable hybridoma clone, designated MH-1, was selected and cultured for mass production of the monoclonal antibody MA-1. MA-1 was purified by affinity chromatography with m-AST as a ligand. The m-AST activity was inhibited uncompetitively by preincubation with MA-1, but preincubation with the polyclonal antibody uncompetitively by preincubation with MA-1, but preincubation with the polyclonal antibody raised in a rabbit resulted in noncompetitive inhibition of the enzyme. These results suggest the usefulness of a monoclonal antibody for studying the mechanism of catalysis. Sandwich enzyme immunoassay methods for m-AST using both polyclonal and monoclonal antibody-coated polystyrene balls were established and permitted the determination of porcine m-AST on the order of 10(-11) and 10(-10)M, respectively.
Animals↗
Evidence for the repetitive domain structure of pig calpastatin as demonstrated by cloning of complementary DNA.
A clone of complementary DNA (cDNA) coding for pig heart calpastatin was isolated using synthetic oligonucleotide probes. The amino acid sequence deduced from the nucleotide sequence revealed the occurrence of a repetitive sequence at the interval of 140 amino acids, substantiating the multidomain structure of calpastatin. A portion of the sequence of 251 amino acid residues predicted for pig heart calpastatin (107 kDa) was found to be identical with that of a peptide fragment derived from pig erythrocyte calpastatin (68 kDa) and sequenced by Edman degradation.
Amino Acid Sequence↗
Ca2+-dependent cysteine proteinase, calpains I and II are not phosphorylated in vivo.
To clarify phosphorylation of calpains I and II in vivo, we purified both calpains concurrently from the [32P] metabolic-labeled human chronic myelogenous leukemia cell line K-562. By Ultragel AcA34 column chromatography, enzymatic activity of calpain I was separated from [32P] radioactivity. Whereas calpain II activity was closely associated with [32P] radioactivity on Ultragel AcA34 and Blue Sepharose CL-6B column chromatographies. By the above purification procedures, calpain I was purified 1300-fold from the crude extract and calpain II was 920-fold from the original sample, respectively. Autoradiographies of purified calpains I and II from [32P] labeled K-562 cells revealed that both calpains were not specifically phosphorylated in vivo. The autophosphorylation in vitro on calpains and modulation of their proteolytic activities reported recently thus may not occur within cells.
Autoradiography↗
Two different molecular species of pig calpastatin. Structural and functional relationship between 107 kDa and 68 kDa molecules.
Calpastatin, the inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase), is known to be widely distributed in mammalian and avian cells. Two different molecular species of calpastatin were isolated and purified to homogeneity from pig heart muscle and from pig erythrocytes, and shown to be of 107 kDa and 68 kDa respectively on SDS/polyacrylamide-gel electrophoresis. Both calpastatins had very similar amino acid compositions when expressed as mol per cent of the residues, differed by only 0.1 pH unit in their isoelectric points, and showed immunological cross-reactivity. One molecule of the 107 kDa species could bind approx. 8 calpain molecules, whereas the 68 kDa inhibitor could bind approx. 5 calpain molecules. These findings suggest similar protein structures of the 107 kDa and 68 kDa calpastatins, each being composed of extended multidomains, with unit inhibitor domains aligned along the polypeptide chain of the molecule. The present study does not conclude, however, whether or not the 68 kDa calpastatin found in erythrocytes is a derived product from the 107 kDa species, which is present as such in heart muscle.
Amino Acids↗
Immunogold electron-microscopic localisation of calpain I in skeletal muscle of rats.
By using a double-affinity-purified first antibody and colloidal gold-conjugated second antibody, it is shown that calpain I (a cysteine proteinase activated by micromolar concentrations of Ca2+) has a predominant intracellular location in the I-band region of the extensor digitorum longus (EDL) muscle of the rat, but is not exclusively associated with the Z-line.
Animals↗
Inactivation of calpain I and calpain II by specificity-oriented tripeptidyl chloromethyl ketones.
Three new tripeptidyl chloromethyl ketones, Leu-Leu-XCH2Cl, with X representing Phe, Tyr, or Lys, were synthesized and their potencies to inactivate calpains I and II were compared. They were designed to fulfil the specificity requirement of calpains established recently. When compared in terms of the dose for 50% inactivation, Leu-Leu-PheCH2Cl was the strongest inactivator, being 500-600 times more effective than tosyl-PheCH2Cl and 5-14 times more than N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64). The potency toward calpain, either I or II, decreased in the order Phe greater than Tyr greater than Lys derivatives greater than E-64, whereas that toward papain was E-64 greater than Lys greater than Phe greater than Tyr derivatives. From the determined kinetic parameters, the Phe derivative was 18.3 and 16.6 times more effective than E-64 on calpains I and II, respectively. Likewise, the rate of the alkylation reaction by these chloromethyl ketones with calpain I was 2-4 times greater than that with calpain II. Leu-Leu-PheCH2Cl and its N-dansylated product should be useful for highly selective affinity labeling of calpains I and II.
Alkylation↗
Biochemical demonstration and immunohistochemical localization of calpain in human skin.
The biochemical properties and immunohistochemical localization of calpain, a Ca++-dependent, intracellular, nonlysosomal cysteine proteinase was examined in human skin. Human epidermal calpain I was fractionated on a DEAE-cellulose column and was found to be half-maximally activated at 3.5 microM free Ca++ and fully activated at 10 microM Ca++ as measured by casein hydrolysis. Immunoelectrophoretic blotting of calpain revealed only a single band of Mr 83,000, when the blot was made with affinity-purified anti-calpain I heavy subunit IgG. Immunohistochemical staining of normal human epidermis showed that calpain I was localized in the cytoplasm of keratinocytes in the mid to upper epidermis but not in the basal cells. In untreated psoriatic epidermis, the deposition of this proteinase was visualized weakly just beneath the stratum corneum. However, remarkable staining was observed after photochemotherapy of topical psoralen plus long-wave UV irradiation. Whether the photochemotherapy induced a quantitative increase in the amount of calpain or merely made calpain more stainable by altering the membrane remains unknown.
Antibody Specificity↗
Reversed distribution of calpains and calpastatin in human pituitary gland and selective localization of calpastatin in adrenocorticotropin-producing cells as demonstrated by immunohistochemistry.
The immunohistochemical distribution of Ca2+-dependent cysteine proteinases (calpains I and II) and their endogenous inhibitor calpastatin in normal and adenomatous human pituitary tissue was studied using specific antibodies. The distributions of calpain and calpastatin were dissimilar in human pituitary gland, i.e. ACTH-immunoreactive cells were strongly positive for calpastatin and negative for calpains. PRL-, GH-, FSH-, and TSH-producing cells were negative for calpastatin, but moderately positive for calpains, especially for calpain II, the high Ca2+-requiring form of the enzyme. Similar results were found in pituitary adenoma tissue. These findings indicate that each type of cells producing a specific hormone is equipped with a different balance of the enzyme-inhibitor system involved in the Ca2+-dependent degradation of intracellular proteins.
Adenoma↗