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Biomedical subjects

A Baici

Publications and source records attributed to A Baici.

At least 55 records · Page 3Linked to original sources
PubMed1984

Interaction of the human leukocyte proteinases elastase and cathepsin G with gold, silver and copper compounds.

Gold thiomalate and the corresponding silver and copper derivatives were investigated as inhibitors of the human leukocyte proteinases elastase and cathepsin G. The kinetic inhibition mechanism for gold- and silver thiomalate is of the hyperbolic non-competitive type with both enzymes and the inhibitory efficiency of the metals increases in the order Cu less than Ag less than Au. On the contrary, D-penicillamine derivatives of the three metals do not influence at all the activity of the two proteinases. Although gold thiomalate is the most efficient of the investigated metal compounds (Ki = 33 microM and 25 microM for elastase and cathepsin G, respectively), the hyperbolic nature of the inhibition imposes a serious limit to its practical usefulness since the maximum inhibitory action on both enzymes is about 40%. We suggest that, in order to act as inhibitor, a copper, silver or gold compound must be able to easily transfer the metal to the enzyme.

Cathepsin G↗
PubMed1984

Kinetics of the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis.

The rate constants for the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis were determined by using a pre-steady-state kinetic approach. kon and koff for complex-formation and dissociation were 1 X 10(6)M-1 X S-1 and 8 X 10(-4)S-1 respectively. Ki was calculated as the ratio koff/kon = 8 X 10(-10)M, the binding of eglin to elastase was reversible and the inhibition mechanism was of the fully competitive type. The mechanistic properties of the system and the biological significance of the rate constants are discussed.

Animals↗
PubMed1984

Cathepsin B-like proteinase as a marker for metastatic tumor cell variants.

Serial transplantation of a spontaneous BDX rat tumor, classified as an anaplastic sarcoma, gives rise to two variants; a rapidly growing nonmetastatic line (AS) and a slowly growing, invasive, and highly metastatic variant (ASML). The availability of two cell lines of the same origin but with markedly differing metastatic potential offers an ideal model for the identification of the cellular properties involved in invasive and/or metastatic behavior. The present work focuses on the pattern of various proteinases in the two tumor cell variants. The findings disclosed one major consistent difference which relates to a cathepsin B-like cysteine proteinase. The metastatic ASML variant manifests exceedingly high intracellular cathepsin B-like activity; in the nonmetastatic AS variant, the activity of this proteinase is significantly lower. Other proteinases, in particular elastase-like, chymotrypsin-like, collagenase-like enzymes and plasminogen activator, showed low, essentially comparable activity patterns. Thus, cathepsin B-like proteinase is a marker enzyme of the metastatic ASML tumor cell variant.

Animals↗
PubMed1984

Extracellular cysteine proteinase and collagenase activities as a consequence of tumor-host interaction in the rabbit V2 carcinoma.

Specimens of the rabbit V2 carcinoma were maintained in organ culture to study the secretion of proteinases. Elastase-like, chymotrypsin-like, plasminogen activator-like, cathepsin B-like and collagenase activities were assayed with sensitive fluorimetric techniques. Of these enzymes, the only activities that were secreted in considerable amounts in primary cultures of tumor tissue were collagenase and a cysteine proteinase resembling cathepsin B. Co-cultures of intraperitoneally grown tumor and normal subcutaneous tissue of the rabbit resulted in significantly higher production of the cysteine proteinase and collagenase compared to the sum of the activities of the separate tissues. Explants of subcutaneous tissue of tumor-bearing rabbits secreted significantly more cysteine proteinase and collagenase than explants from normal animals. Explants from normal subcutaneous tissue stimulated with tumor-conditioned culture medium secreted both enzymes in higher amounts compared to the controls. The cysteine proteinase was similar in some properties to rabbit liver cathepsin B, but the enzyme from the tumor-host system showed a remarkable stability to a moderately alkaline pH. We suggest that a diffusible factor, derived from the tumor or immigrated cells, promotes an increased synthesis and secretion of collagenase and cysteine proteinase in the host, and that both enzymes may play cooperative roles during invasion of the surrounding tissues by the V2 carcinoma.

Animals↗
PubMed1982

Methodologic problems encountered in the assay of proteinases in Lewis lung carcinoma, a mouse metastasizing tumor.

The proteolytic activity in homogenates and extracts of subcellular fractions prepared from subcutaneous Lewis lung carcinoma was determined using proteins and synthetic peptides as substrates. The presence of cathepsin D, plasminogen activator, cathepsin B-, cathepsin G- and elastase-like enzymes was observed. No difference was revealed between the proteolytic activity in homogenates of Lewis lung carcinoma, at the growth stage examined, and in homogenates of normal lung. High specific activities were found in the lysosomal extract, whereas decreasing activities were found in the nuclear extract, the homogenate and the postlysosomal mitochondrial supernatant; no active or trypsin-activatable collagenase activity was detected. The presence in the tumor tissue of these enzymatic activities is in agreement with their proposed role in the process of metastasis. The lack of differences between homogenates of tumor and normal lung tissue suggests that the use of whole cells is required to selectively study tumor proteinases specifically involved in tumor malignancy.

Animals↗
PubMed1982

Action of collagenase and elastase from human polymorphonuclear leukocytes on human articular cartilage.

Collagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen. Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate. Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage. The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.

Cartilage, Articular↗
PubMed1982

Cathepsin G from human polymorphonuclear leukocytes cleaves human IgM.

Cathepsin G, the chymotrypsin-like enzyme from human polymorphonuclear leukocytes, cleaves human IgM and produces two major fragments that closely resemble those released by leukocyte elastase digestion of IgM. An F(ab)2 mu-like fragment, mol. wt 140,000, retains some reactivity with an anti-Fc mu antiserum and is antigenically deficient with respect to the IgM subunit. The other fragment is an Fab mu-like product with a mol. wt of 54,000. Both cathepsin G fragments are indistinguishable from the elastase counterparts by immunochemical analysis. An Fc mu fragment could not be recovered. The kinetic course of the cleavage shows that cathepsin G produces Fab mu fragments at a higher rate than F(ab)2 mu, whereas the contrary is valid for elastase. Beside the two major fragments and low mol. wt peptides, cathepsin G releases also a product with the same mol. wt and immunological reactivity as the IgM subunit. The biological significance of the interaction between IgM and leukocyte proteinases is discussed.

Antibodies, Monoclonal↗
PubMed1982

Cleavage of the four human IgG subclasses with cathepsin G.

Cathepsin G, the chymotrypsin-like serine proteinase from human polymorphonuclear leucocytes, cleaves human IgG. The relative susceptibilities of the four IgG subclasses to the action of this enzyme were studied kinetically and showed the following decreasing order of susceptibility: IgG3 much greater than IgG4 greater than IgG1 greater than IgG2. IgG1 and IgG2 produced primarily F(ab')2 and traces of Fc-related fragments. IgG4 gave rise to both Fab and F(ab')2 as major products, and small amounts of an Fc-related fragment were detected. The cleavage of IgG3 produced various fragments, depending on the experimental conditions: The primary fragments were Fab and Fch (Fc covalently joined to the extended hinge-region polypeptide of IgG3) and an intermediate Fab-Fch species. Both Fab and Fch were further degraded by cathepsin G. Fch was gradually split, giving rise to three subfragments that were finally degraded to dialysable peptides. The enzyme further cleaved the Fab fragment in the heavy-chain portion and released a polypeptide probably representing the VH domain.

Animals↗
PubMed1982

The slow, tight-binding inhibition of cathepsin B by leupeptin. A hysteretic effect.

Leupeptin was found to be a slow, tight-binding inhibitor of cathepsin B from human spleen and rabbit liver. During the enzyme-catalyzed reaction in the presence of inhibitor a concentration-dependent transient state, lasting several minutes, preceded the attainment of the steady state and was characterized by a concave upward or a concave downward lag phase depending on whether the enzyme had been preincubated with the inhibitor or not, respectively. From the pre-steady-state phase of the curves both k on and k off for the formation of the enzyme-inhibitor complex could be calculated. Ki, as the ratio k off/k on, was in good agreement with the inhibition constant obtained using a steady-state treatment. k on was 1.8 X 10(5) M-1 s-1 and 2.0 X 10(5) M-1 s-1 for the human and rabbit enzyme, respectively and the slowness of the binding process fitted into the general concept of enzyme hysteresis. The activation of the essential cysteine residue of cathepsin B by dithiothreitol was also a very slow process characterized by a second-order rate constant of 4.1 M-1 s-1. The kinetic features of leupeptin binding allow the prediction of the possible efficiency of this inhibitor on cathepsin B in vivo. It is shown that in order for leupeptin to be a physiologically significant inhibitor of cathepsin B, its concentration at the target site must exceed 10 microM, at least. This contrasts with the predictions drawn from the value of Ki (approximately 5 nM), which would suggest an effective inhibition of the enzyme already at a concentration of 0.05 microM.

Animals↗
PubMed1981

Production of agglutinators and rheumatoid factors in plasma cells of rheumatoid and nonrheumatoid synovial tissues.

Immunohistochemical studies were performed in synovial tissues from 40 patients with rheumatoid arthritis (RA), 9 with juvenile rheumatoid arthritis (JRA), 7 with psoriatic arthritis, and 4 with various rheumatic diseases. Overall synthesis of IgG- and/or IgM-rheumatoid factor (RF) was found in all patients with seropositive RA and JRA, in 75% of patients with seronegative RA, and in 1 patient with psoriatic arthritis. Agglutinator producing cells were found in 77% of the samples from seropositive RA and in 44% and 56% from seronegative RA and JRA patients, respectively. The percentage of IgG plasma cells synthesizing one or more of the 5 types of agglutinators studied was approximately 10% of plasma cells synthesizing IgG-RF. Intercellular and intracellular immune complex deposits were also found in patients with seropositive and seronegative RA and JRA. These findings suggest that synthesis of agglutinators by synovial tissue plasma cells of RA and JRA patients is a distinct--but definitely less prominent--function than that of RF synthesis.

Agglutinins↗
PubMed1981

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes.

Using a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C3, alpha 1-PI and rarely alpha 2-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.

Adult↗
PubMed1981

The specific velocity plot. A graphical method for determining inhibition parameters for both linear and hyperbolic enzyme inhibitors.

A new method is described for plotting kinetic results for inhibited enzymatic reactions. The plot is particularly designed for the analysis of hyperbolic, mixed-type inhibitors, but can be applied to any other linear system. The method consists of plotting experimental data in a normalized way, as v0/vi versus the specific velocity sigma/(1 + sigma), where v0/vi represents the ratio of the initial velocities for the non-inhibited and inhibited reactions at a given substrate concentration [S], and sigma is the [S]/Km ratio. The procedure provides a simple way of determining the inhibition constants Ki and Ki', i.e, the dissociation constants of the EI and ESI complexes, respectively, and the velocity saturation values.

Enzyme Inhibitors↗
PubMed1981

Histochemical localization of cathepsin B at the invasion front of the rabbit V2 carcinoma.

To clarify the role of cathepsin B in tumor invasion, the enzyme was visualized in tissue frozen sections of the subcutaneously growing rabbit V2 carcinoma. Localization of cathepsin B was achieved by immunofluorescent staining and by enzyme histochemistry. For the former approach, a sheep antiserum was raised against purified cathepsin B from rabbit liver. The antibodies, isolated by immunoadsorption, reacted monospecifically with rabbit liver cathepsin B in Ouchterlony double diffusion and in immunoelectrophoresis. In the enzyme histochemical assay, Z-Ala-Arg-Arg-methoxynaphtylamide was used as fluorogenic substrate and nitrosalicylaldehyde as coupling agent. With both methods, cathepsin B was found to be localized within fibroblasts and leukocytes assembled at the tumor invasion front. In addition, immunofluorescent staining demonstrated the occurrence of the enzyme in the extracellular matrix surrounding tumor cell clusters. Carcinoma cells always remained unstained. The conclusion is drawn that cathepsin B is chiefly produced by host cells which are stimulated to increase synthesis and to release the enzyme under the influence of the tumor. A dual function can be ascribed to cathepsin B concentrated in the vicinity of the tumor: it operates intracellularly (in host cells) through degradation of endocytosed protein and extracellularly through activation of collagenase. The resulting lytic action on host structures appears to be a prerequisite for local spread of the V2 carcinoma.

Animals↗
PubMed1980

Kinetics of the different susceptibilities of the four human immunoglobulin G subclasses to proteolysis by human lysosomal elastase.

Human lysosomal elastase cleaves human monoclonal IgG into components that closely resemble the fragments produced by papain digestion. IgG1 produced Fab, Fc and Fab-Fc fragments; cleavage of IgG2 produced F(ab)2, Fab-Fc, Fab and Fc fragments; IgG3 gave rise to almost pure Fab and Fch (Fc covalently joined to the extended hinge region polypeptide of IgG3), and from IgG4, F(ab)2, Fab and Fc fragments were recovered. The relative susceptibilities of the four human IgG subclasses to proteolytic attack by elastase were studied kinetically and showed the following decreasing order of susceptibility: IgG3 > IgG1 > IgG2 > IgG4. The Fab fragment from papain digestion of IgG1 and the corresponding fragment from elastase digestion showed indistinguishable molecular weights and immunochemical identity.

Chromatography, Gel↗
PubMed1980

Partial purification and characterization of the high molecular weight latent collagenase from human leukocytes.

Two latent collagenases whose apparent molecular weights were ca. 150 000 and 60 000 have been detected in human leukocytes and the partial purification of the high molecular weight component was accomplished by the following steps: acetone precipitation, ammonium sulphate fractionation, gel filtration on Sephacryl S-200, chromatography on DEAE-Sepharose, and a final gel filtration on Sephacryl S-200. After activation by an activator extracted from human rheumatoid synovial fluid, the enzyme was able to cleave collagen into the classical 1/4 and 3/4 fragments, and was inhibited by chelating agents and other typical collagenase inhibitors.

Arthritis, Rheumatoid↗