Binding of the cysteine proteinases papain and cathepsin B-like to coated laminin: use of synthetic peptides from laminin and from the laminin binding region of the beta 1 integrin subunit to characterize the binding site.

Cysteine proteinases of the papain superfamily, i.e., papain and cathepsin B-like proteinase, were found to be able to bind to laminin-coated wells.

Expression of collagenase/gelatinase activity from basement-membrane fibronectin--isolation after limited proteolysis of a bovine lens capsule and molecular definition of this thiol-dependent zinc metalloproteinase.

We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra-domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37 degrees C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7-methoxycoumarin-4-yl)-acetylprolylleucylglycylleucyl-[3-(2,4-di nitrophenyl)-L-2,3-diaminopropionyl]-alanylarginylamide.

Competition between plasminogen and procathepsin B as a probe to demonstrate the in vitro activation of procathepsin B by the tissue plasminogen activator.

The tissue plasminogen activator (tPA) was found to activate in vitro the procathepsin B purified from malignant ascitic fluids. This activation was time and dose dependent, and was associated with the processing of procathepsin B. The present study shows that tPA is a fast activator of procathepsin B in a neutral pH range, such that generation of cathepsin B activity and processing of procathepsin B are achieved after a 5-min incubation time at 37 degrees C, pH 7.

Immunopurification and characterization of a collagenase/gelatinase domain issued from basement membrane fibronectin.

The proteolytic potential of cellular fibronectin fragments issued from a basement membrane hydrolysate was investigated. Three different gelatinase activities (47, 43 and 37 kDa), located by gelatin zymography, were isolated using successively heparin-agarose, gelatin-agarose and immunopurification with polyclonal antibodies directed against bovine plasma fibronectin. These fragments were also characterized using a monoclonal antibody directed against the extra-domain EDA of cellular fibronectin as a probe.

"In vitro" study of basement membrane degradation by the cysteine proteinases, cathepsins B, B-like and L. Digestion of collagen IV, laminin, fibronectin, and release of gelatinase activities from basement membrane fibronectin.

We have studied the soluble fragments obtained from bovine lens capsules after digestion by the cysteine proteinases cathepsins B, B-like and L. These proteinases liberated collagen IV, laminin and fibronectin fragments, as shown by immunoblotting. Sodium dodecyl sulfate treatment of digested capsules gave a soluble material used for subsequent fractionation and immunochemical study.

In vitro activation of pro-cathepsin B by three serine proteinases: leucocyte elastase, cathepsin G, and the urokinase-type plasminogen activator.

In vitro activation of pro-cathepsin B purified from ascitic fluid of ovarian carcinomas by serine proteinases was studied. Both elastase and cathepsin G from human leucocytes were found to be activators, on the basis of generation of cathepsin B activity and processing of the precursor. These results represent a new cooperative pathway between cancer cells and host cells.

[Quantitative binding of cysteine-proteinases to a basement membrane: role in their digestion during tumor invasion].

Binding of cysteine-proteinases of the papain-superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. The basement membrane used for these experiments is the bovine lens capsule (weight approximately 50 mg). Papain inactivated by E64 was used in competition experiments, that made it possible to obtain the equilibrium constant, Kd and the number of substrate sites per capsule, n.

Quantitative study of the binding of cysteine proteinases to basement membranes.

Binding of cysteine proteinases of the papain superfamily (papain and cathepsins B, B-like and L) to basement membranes was studied by using the enzymatic activity of these proteinases against their specific fluorogenic substrates. Papain inactivated by E64 was used for Kd determination by competition experiments. The binding was characterized using the following parameters, the equilibrium constant, Kd, and the number of substrate sites, n, values of which were in the range of 10(-7) M and 10(12), respectively.

High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver.

A high-performance liquid chromatographic procedure for the isolation of the three cysteine proteinases, namely cathepsins B, H and L, is described. The method is based on the following four steps. (1) A classical AcA 44 gel permeation separation with a 30-70% ammonium sulphate fraction from the human liver homogenate is used to remove the non-enzymic high-molecular-mass components.

[Digestion in vitro of basal membrane, bovine lens capsule, by human liver lysosome cathepsins B, H and L and ascitic fluid tumor cathepsin B from ovarian adenocarcinoma].

Lysosomal cysteine proteinases, cathepsins B, H and L, and tumor cathepsin-B-like protease, are capable of digesting bovine lens capsule, a typical basement membrane, in vitro. Cathepsins L and H proved most active in the pH range 5.0-7.

"In vitro" digestion of intact bovine lens capsules by four human lysosomal cysteine-proteinases.

We have examined the biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) by human liver cathepsins B, H and L and the cathepsin B-like proteinase from malignant ascitic fluid. This study was carried out using two different methods: The first strategy was to follow the liberation of soluble proteins and peptides as a function of time at different pHs. Then the digestion products were characterized, as collagen IV, fibronectin and laminin fragments, using monospecific polyclonal antibodies and a quantitative dot-blot analysis.

The glycosylation state of the precursors of the cathepsin B-like proteinase from human malignant ascitic fluid: possible implication in the secretory pathway of these proenzymes.

We have investigated the structure of the carbohydrate moiety of the precursors of the cathepsin B-like proteinase (PCBt). The largest precursor has an apparent molecular size (Mr) of 45-47 Kd and it contains 3 N-linked oligosaccharide chains which were Endo beta N acetylglucosaminidase H (Endo H)-resistant forms. On the other hand, these chains were sequentially removed by Endo beta N acetylglucosaminidase F (Endo F).

Immunohistochemical and biochemical study of a cathepsin B-like proteinase in human colonic cancers.

An immunohistological study was carried out on 51 human colorectal adenocarcinomas and eight samples of histologically normal colonic mucosa removed far from tumors, using anti-rabbit cathepsin B and anti-human cathepsin B immunoglobulins. Positive reactions were obtained on tumor cells and macrophage-like cells. However, as these immunoglobulins could not discriminate between cathepsin B and cathepsin B-like proteinases, and as they cross-reacted with cathepsins H and L, a partial characterization of the proteinase activities was performed in order to identify the type of enzyme present in the positive cells.

Purification and characterization of two different precursor forms of the cathepsin B-like proteinase from human malignant ascitic fluid.

We have purified two different precursors of a cathepsin B-like proteinase (PCBT) from malignant ascitic fluid. The molecular mass of these proteins were 45-47 kDa and 36 kDa, respectively. This report is the first which shows cathepsin-B precursors as purified proteins.

[Regulation of neoplasm-specific cathepsin B by cysteine-protease inhibitors present in cancerous exudates].

The Cathepsin B-like proteinase, a secretory form of lysosomal cathepsin B, is present in some cancerous exudates (i.e. pleural and ascitic fluids).