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E Krepela

Publications and source records attributed to E Krepela.

35 records · Page 2Linked to original sources
PubMed1989

A kinetic fluorometric assay of dipeptidyl peptidase IV in viable human blood mononuclear cells.

A continuous-rate fluorometric assay of dipeptidyl peptidase IV (DP-IV) in viable human blood mononuclear cells using 7-(L-glycyl-L-prolylamido)-4-methylcoumarin as the substrate is described. The assay method is accurate, rapid, and highly sensitive for measuring the level of cell-surface bound DP-IV activity in suspension of blood mononuclear cells, as well as of other viable cells bearing this enzyme. We believe that the kinetic assay is suitable for studying the regulation of expression and the role of plasma membrane-bound DP-IV on the cellular level.

Cell Separation↗
PubMed1989

Cathepsin B in human breast tumor tissue and cancer cells.

The cysteine proteinase cathepsin B (EC 3.4.22.1) has been proposed to play an important role in the proteolytic mechanism of the ability of breast cancer cells to invade into and through normal tissues during metastasis. In this study, activity of cathepsin B was measured with a fluorometric microtiter plate assay in human breast tumors as well as in mammary gland dysplasias and in four human breast cancer cell lines (BT-20, MDA-MB-231, PMC42 and T47D). It was found that primary breast carcinomas and cystosarcomas phyllodes contain significantly higher levels of cathepsin B activity than mammary dysplasias; the activity of cathepsin B in cystosarcomas phyllodes was comparable with that in breast carcinomas. The enzyme from breast carcinoma tissue exhibited properties of a mature form of cathepsin B. All investigated breast cancer cell lines display positive cytochemical staining for cathepsin B activity with granular pattern of distribution of the final reaction product. Biochemically, the breast cancer cell lines differed significantly from each other in the level of cathepsin B activity decreasing in the following order: T47D, PMC42, MDA-MB-231 and BT-20.

Breast Neoplasms↗
PubMed1989

Cathepsin B in cells of two rat sarcomas with different rates of spontaneous metastasis.

The cysteine proteinase cathepsin B (EC 3.4.22.1) is believed to take part in biochemical processes underlying tumor metastasis. In the present study, the cellular localization, intracellular levels and extracellular release of cathepsin B activity were examined in vitro in cells of two rat sarcoma variants, LW13K2 and RPS, differing in their capacity to metastasize spontaneously to the lung of syngeneic LEW/CUB rats. The LW13K2 sarcoma metastasizes rarely, whereas the LW13K2-derived RPS variant produces a metastasis incidence of above 50%. Using fluorescent cytochemical staining, microgranular reaction centers of cathepsin B were observed in the cell cytoplasm, in some cellular processes, with apical localization in some of them, as well as at the extreme cell periphery in cells of both sarcoma variants. The appearance of this distribution of cathepsin B activity was delayed in the RPS variant. Biochemically, the intracellular level of cathepsin B activity was significantly higher in homogenates of LW13K2 cells than RPS cells. In contrast to the intracellular enzyme activity, RPS cells cultured in serum-free medium at pH 6.5 released a substantially higher amount of cathepsin B activity than LW13K2 cells into the extracellular environment; at pH 7.4 the initially higher release of cathepsin B activity from RPS cells later equalized with that from LW13K2 cells. Taken together, the results indicate that changes of pericellular pH can modulate the extracellular release of cathepsin B in both sarcoma cell variants and suggest that the rate of cathepsin B release under conditions of mildly acid pericellular pH could be related to the incidence of metastases observed in these rat sarcoma variants. Total intracellular cathepsin B activity did not exhibit positive correlation with the metastatic potential of the studied rat sarcoma variants.

Animals↗
PubMed1987

A latent form of cathepsin B in pleural effusions. I. Characterization of the enzyme in breast cancer patients.

A latent cysteine proteinase has been found in the pleural effusion fluid of patients with breast cancer. It can be converted by pepsin to an active form, the properties of which, including the pH optimum, pH stability, substrate specificity, and sensitivity to various proteinase inhibitors, were found to be closely related to those of cathepsin B. Unlike the pepsin-generated enzyme, which was rapidly inactivated above pH 7.0, the latent enzyme showed substantially higher stability in the region around and above neutral pH. The apparent Mr values of the latent and pepsin-generated enzyme forms were approximately 45,000 and 32,000, respectively. Both enzyme forms exhibited heterogeneous binding affinity to concanavalin A-Sepharose 4B. Altogether, our results demonstrate that a latent cathepsin B form occurs in vivo in pleural effusions of breast cancer patients.

Breast Neoplasms↗
PubMed1987

Cytochemical and biochemical evidence of cathepsin B in malignant, transformed and normal breast epithelial cells.

Human breast cancer cell lines, as well as transformed mammary epithelial cells (HBL-100) and growth-stimulated normal breast epithelial cells showed positive cytochemical reaction with the proteinase substrate 2-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methoxynapht halene, in the presence of 5-nitrosalicylaldehyde. The reaction product, small fluorescent granules, was distributed throughout the cytoplasm, in the perinuclear zone, in some cytoplasmic projections, and at the cell surface. Using a panel of various proteinase inhibitors, we found that the formation of the reaction product was an enzymic function of a cysteine proteinase. Using the substrate 7-(N-benzyloxycarbonyl-L-arginyl-L-arginylamido)-4-methylcoumarin, we evaluated some biochemical properties of the cysteine proteinase, including pH-activity profile, pH stability, apparent relative molecular mass and sensitivity toward various proteinase inhibitors. We found that the proteinase from the studied breast epithelial cells exhibited characteristics of a mature form of cathepsin B. Taken together, the cytochemical and biochemical data provide evidence that human breast epithelial cells of cancer origin, as well as in the transformed or growth-stimulated state express active cathepsin B and compartmentalize it into specific subcellular sites.

Breast↗
PubMed1987

Clinical experience with the testing of serum proteinase activity by cathepsin B-like sensitive amino acid derivatives of 7-amino-4-methylcoumarine.

The proteolytic activity of serum against two synthetic polypeptide substrates conjugated with 7-amino-4-methylcoumarine which are known to be highly specific substrates for lysosomal cathepsin B-like cysteine proteinase, was studied in normal individuals and in patients with breast and colorectal cancer. The results showed that both substrates are cleaved in different ways and their hydrolysis is very poorly sensitive to inhibitors of cysteine proteinases. On the other hand, cleavage is intensively inhibited by disodium-EDTA which is known as an activator of cysteine proteinases. This indicates that lysosomal conditions are quite different from serum which is an unstable mixture of various enzymes and their substrates. It was also demonstrated that hydrolysis of the tested substrates is not probably realized by cysteine proteinases but it can be performed by cooperation of other serum proteinases. Moreover, our results confirmed that serum activity of these enzymes can be significantly higher in patients with breast cancer, particularly with metastasis, than in healthy women. A slight insignificant increase was observed also in patients with colorectal cancer.

Adult↗
PubMed1985

Changes in tissue and serum activity of cathepsin B-like cysteine proteinase during colorectal carcinogenesis by 1,2-dimethylhydrazine in mice.

The activity of cathepsin B-like cysteine proteinase in the mice colon was studied during carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride (DMH). Before starting the treatment with DMH, the activity of the observed enzyme was very low in the colorectal area but the activity in the serum was rather high. In the course of carcinogenesis, the local activity markedly increased in the stroma equally as in the cells of developing tumors while the serum activity slightly decreased. Following the total ovariectomy, no significant changes in the local activity or in the serum activity were found. Possible causes of these variations are discussed.

1,2-Dimethylhydrazine↗
PubMed1983

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates.

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.

Adult↗
PubMed1983

Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum.

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.

Chromatography, Gel↗