Shizuko Takahashi

Collagen loss in the stunned myocardium.

Background This study was performed to biochemically assess and quantify the previously observed ultrastructural alterations in the collagen matrix of stunned myocardium. Methods and Results The stunned myocardium was produced in 13 mongrel dogs by a series of 12 coronary artery occlusions of 5 minutes followed by 10-minute reperfusion periods, with a final reperfusion period of 90 minutes. Regional systolic function in the stunned myocardium was 17% of control. Relative end-diastolic length in the stunned region increased up to 8%. There was a nonuniform transmural loss of collagen. Hydroxyproline in the stunned endocardium was not different from control. The stunned midwall and epicardium demonstrated 12.5% (p <0.05) and 14.6% (p <0.005) decreases, respectively. All transmural layers in the stunned myocardium had significant increases in collagenase activity before procollagenase activation, averaging a 73.6% increase (p <0.025). Complete activation of all procollagenase forms with aminophenylmercuric acetate revealed no differences in fully activated collagenase between the stunned and normal regions. The lysosomal enzymes, elastase and cathepsin G, were not different between stunned and normal zone tissue. These results would tend to exclude exogenous sources of protease in the stunned myocardium at the 90-minute final reperfusion time frame. Collagen fibers were isolated from the stunned and normal zone tissue and underwent dansyl chloride reaction. Stunned collagen fibers had 9% greater dansyl labeling, suggesting greater numbers of exposed N-terminal amino acid residues on the fiber and compatible with greater enzymatic cleavage activity on the stunned collagen matrix. Tissue water content was consistently greater in the stunned region compared to the normal: a uniform transmural increase of approximately 1.7%. Conclusions The stunned myocardium is characterized by both systolic dysfunction and diastolic expansion or dilatation. Endogenous procollagenase is activated by the ischemic process leading to degradation of the extracellular matrix. The underlying mechanisms may be relevant in ischemic enlargement of the heart and cardiomyopathy.

A new radioactive assay for enzymes with elastolytic activity using reduced tritiated elastin, the effect of sodium dodecyl sulfate on elastolysis

Abstract A method is described for the assay of elastolytic activity based on the use of an insoluble elastin substrate the aldehydes and cross-links of which have been labeled by reduction with tritiated NaBH 4 . The rate of appearance of radioactivity elastolysis than both the rates of appearance of materials that react with ninhydrin or with the biuret-Folin reagent. Experiments were also perfomed to determine whether the reported enhancement of elastolysis occasioned by pretreatment of the substrate with sodium dodecyl sulfate could be verified using the radioactivity assay, induced the rate of elastolysis was approximately doubled when the labeled substrate was first treated with sodium dodecyl sulfate. Comparison of the rates of liberation to the soluble phase of radioactivity, ninhydrin-reacting material and of peptides reacting with the biuret Folin reagent permits an inference that sodium dodecyl sulfate, in addition to inducing formation of new regions of α helix, may expose the polypeptide regions surrounding the cross-links allowing elastase subsequently to cleave these regions more rapidly.

γ-Glutamyl transpeptidase and glutathione in aging IMR-90 fibroblasts and in differentiating 3T3 L1 preadipocytes

Abstract γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities, and glutathione contents were measured in IMR-90 cells, human embryonic lung diploid fibroblasts, “aging” in culture, and in cultures of 3T3 L1 mouse preadipocytes differentiating to adipocytes. γ-Glutamyl transpeptidase, glutathionase, and phosphateindependent glutaminase activities in IMR-90 cells were about 8-, 8-, and 7-fold higher in “old” cells as compared to the activities in “young” cells. Total and reduced glutathione contents, expressed in relation to either cell number or DNA content, of IMR-90 cells were 3- and 12-fold higher in “young” cells (population doubling level 22) as compared to “old” cells (population doubling level 49). The ratio of reduced to oxidized glutathione was relatively constant in “young” cells over a range of population doubling levels 18–25; however, relative to the “young” cells, the ratio was decreased in “old” cells over a range of population doubling levels 49–55. γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities in 3T3 L1 cells were about 4-, 5-, and 5-fold higher in differentiating cells as compared to the activities in undifferentiating cells. Total glutathione contents in 3T3 L1 cells differentiating to adipocytes were similar to glutathione contents in undifferentiating cells. As the culture differentiated to the extent of 50%, almost all of the glutathione was present as oxidized glutathione. These results suggest that the several enzymatic activities and glutathione contents may be used as markers for aging or differentiation in cells. In the course of these studies, a sensitive fluorometric assay was developed, using l -γ-glutamyl-4-methoxy-2-naphthylamide as substrate.

Immunohistochemical localization of collagenase in hepatic murine schistosomiasis.

It has been previously demonstrated that collagenase activity and collagen synthesis in hepatic granulomas of mice infected with S. mansoni cercariae are maximal 8 weeks after infection; however, total liver collagen content continues to increase. Now the anatomic relationships among collagenase and collagen, granulomas, and hepatic parenchyma in normal mice and in mice infected with S. mansoni are studied. Trypsin-activated collagenase was purified from the media of cultured granuloma explants and anti-collagenase immunoglobulin G was purified from immunized rabbits. The IgG cross-reacted with liver granulomas and active and inactive forms of collagenase, but did not react by immunodiffusion in agar with other neutral proteases or homogenates of schistosome eggs or normal liver. Cryostat sections of liver from normal and infected mice were studied by indirect immunohistochemical methods using fluorescein, rhodamine, and peroxidase labels. Collagenase localization was restricted to areas of collagen deposits in granulomas and hepatic parenchyma. Ultrastructural studies revealed collagenase on the surface of collagen fibers. Hepatocytes of normal mice showed delicate staining at the sinusoidal surface. At all times, immunoreactive collagenase was intimately associated with its substrate, where it presumably initiated collagen degradation. This localization provides a rationale for possible therapeutic approaches to control fibrogenesis through collagenase induction or activation.

Purification of γ-glutamyltransferase of rat kidney by affinity chromatography using concanavalin a conjugated with sepharose 4B

Abstract A procedure is presented for the purification of γ-glutamyltransferase (EC 2.3.2.2) of rat kidney by affinity chromatography using concanavalin A conjugated to Sepharose 4B. The enzyme is eluted from the conjugate by a solution of methyl α- d -mannoside. The enzyme, when bound to concanavalin A-Sepharose, is active in a suspended form, suggesting that the catalytic site is different from the site that binds to concanavalin.

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.

γ-Glutamyl transpeptidase from WI-38 fibroblasts: Purification and active site modification studies

γ-Glutamyl transpeptidase has been purified to homogeneity from WI-38 human fetal lung fibroblasts, following extraction with Triton X-100 in the absence of added proteases. The specific activity of the purified enzyme is 16 units/mg protein at the optimum of pH 8.0. Although this activity value is low, the WI-38 enzyme is very similar to previously described γ-glutamyl transpeptidases in its molecular properties. The native molecule (apparent molecular weight of 82,000) is composed of one light and one heavy subunit (apparent molecular weights of 20,000 and 62,000, respectively). Papain digestion reduces the native molecular weight to an apparent value of 73,000 by proteolysis of the heavy chain. The known active site modifying agent and glutamine analog 6-diazo-5-oxo-l-nor-leucine, completely inactivates the enzyme, coincident with its stoichiometric incorporation into the light subunit. This inactivation is accelerated by maleate and prevented by S-methylglutathione. The WI-38 γ-glutamyl transpeptidase is also inactivated by the fluorescent alkylating agent, 5-iodoacetamidofluorescein. Selective reaction of this reagent with an active site residue is suggested by prevention of the inactivation by S-methylglutathione, the stoichiometric incorporation of the fluorescein moiety, and the loss of one methionine residue per molecule of protein accompanying inactivation.

γ-glutamyltransferase in human diploid fibroblasts and other mammalian cells

Summaryγ-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that γ-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.

Enzymes of the γ-glutamyl, cycle in ‘aging’ WI-38 fibroblasts and in HeLa S3 cells

Abstract γ-Glutamyltransferase ((5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2) activity of WI-38 fibroblasts decreased only slightly in relation to a constant amount of cell-associated protein as the cells were carried in culture serially from middle to late passage numbers leading toward senescence, e.g., from population doubling level 27 through 41. Also, when the enzyme activity was expressed on the basis of a unit number of cells or unit amount of DNA, little change occurred over that range of PDLs. As the culture approached ‘phase-out’, the transferase activity rose sharply regardless of how the activity was expressed. The possibility is considered that the large increase in activity could be a reflection of a significant increase in size of cells and therefore changes in the membranes where the transferase is located. The occurrence of other enzymes of the ‘gg-glutamyl cycle’ in WI-38 and HeLa S 3 cells also was demonstrated. These included γ-glutamylcyclotransferase ((γ- l -glutamyl)- l -amino-acid γ-glutamyltransferase (cyclizing), EC 2.3.2.4) and 5-oxoprolinase, whose actitivies showed no large increase comparable to that of the γ-glutamyltransferase, as the culture approached ‘phase-out’.

Elastolytic activities of Clostridium histolyticum.

Abstract By combinations of column chromatography, four fractions with elastolytic activity have been prepared from medium in which Clostridium , histolyticum has grown. One of these, Fraction C, is of very high specific activity and in fairly pure form. Three fractions in particular have been characterized. Like other elastases they also exert general protease activity, and can digest casein. Their actions against elastin are DIPF-sensitive and EDTA-insensitive. Their actions against casein are EDTA-sensitive and DIPF-in-sensitive. In crude form they can digest the peptide substrate developed by Visser and Blout for pancreatic elastase. The highly active clostridial Fraction C yields larger peptides with elastin than does pancreatic elastase, and eventually may be used in conjunction with that enzyme in sequence studies of elastin.