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Biomedical subjects

J Saklatvala

Publications and source records attributed to J Saklatvala.

99 records · Page 6Linked to original sources
PubMed1981

Isolation and characterization of tryase, a serine proteinase from rat liver.

1. A new serine proteinase, tryase, was isolated from the membrane fraction of a post-nuclear supernatant of rat liver homogenate. The enzyme was solubilized with 1 M-MgCl2 and purified to homogeneity by DEAE-cellulose chromatography and affinity chromatography with soya-bean trypsin inhibitor linked to Sepharose 4B. 2. The enzyme was identified on sodium dodecyl sulphate/polyacrylamide gels by reaction with radiolabelled di-isopropyl phosphorofluoridate. Unreduced its molecular weight was 32 500, reduced it was 28 000. 3. The enzyme readily hydrolysed azocasein and tripeptide nitroanilide substrates with an arginine or lysine residue adjacent to the leaving group. D-Pro-Phe-Arg-NPhNO2 was used routinely (Km = 0.25 mM). Tryase showed little activity on blocked arginine esters or amides. 4. It was inhibited by di-isopropyl phosphorofluoridate, benzamidine, aprotinin, soya-bean and lima-bean trypsin inhibitors, Ile-Leu-Arg-CH2Cl and Phe-Ala-Arg-CH2Cl. It was not inhibited by Tos-Lys-CH2Cl. 5. Subcellular-fractionation studies showed that tryase was associated with particles similar in their sedimentation properties to lysosomes, but, since it was not present in tritosomes, it was not in the classical lysosome. 6. Rat liver contained other neutral proteinases; one of these was a serine proteinase with an apparent molecular weight of 90 000 on gel chromatography.

Animals↗
PubMed1980

Identification of proteinases in rheumatoid synovium. Detection of leukocyte elastase cathepsin G and another serine proteinase.

Extracts of rheumatoid synovial tissue obtained at surgical synovectomy contained neutral proteinases as well as cathepsin D. The neutral proteinase activity was particle-bound but could be solubilized by 1 M MgCl2. About half of the solubilized activity adsorbed to aproptinin-Sepharose at pH 7.5 and was desorbed at pH 3.3. This activity was shown to be due to leukocyte elastase and cathepsin G by enzymological and immunological criteria. The neutral proteinase activity that did not adsorb to aprotinin-Sepharose was not due to elastase or cathepsin G. It was able to hydrolyse proteoglycan and was inhibited by diisopropylfluorophosphate, soybean and lima bean trypsin inhibitors. It was, therefore, a serine proteinase. Its inhibition characteristics were different from those of plasmin, kallikrein or thrombin. All of the neutral proteinase activity of synovial extracts was attributable to serine proteinases, no evidence of metallo-proteinases was found. The possible role of the neutral proteinases in the degradation of the matrix of cartilage is discussed. A simple procedure for purifying leukocyte elastase and cathepsin G is described as well as the raising of specific antisera to these enzymes.

Aprotinin↗
PubMed1979

A cartilage catabolic factor from synovium.

Porcine synovium in organ culture produces a factor that causes chondrocytes to degrade their matrix. A quantitative assay for the factor, for which the cartilage of bovine nasal septum is used, is described. Evidence is presented that the catabolic factor is a protein.

Animals↗
PubMed1977

Hydrolysis of the elastase substrate succinyltrialanine nitroanilide by a metal-dependent enzyme in rheumatoid synovial fluid.

A new type of enzyme hydrolyzing the elastase substrate succinyl-L-alanyl-L-alanine-4-nitroanilide has been found in cell-free rheuma todi synovial fluid. Normal plasma and osteoarthritic synovial fluid contained relatively little enzyme. The pH optimum was 8.0. Unexpectedly, the enzyme activity was not due to leukocyte elastase or any proteinase bound to alpha2-macroglobulin. The enzyme activity was metal-dependent being inhibited by chelating agents but not by di-isopropylfluorophos phate or thiol-blocking reagents. Gel chromatography showed the enzyme activity was associated with material of high molecular weight. On Sepharose 4B chromatography two-thirds of the activity eluted in the void volume and one-third in a position of about 106 mol wt. Utracentrifugation showed that both components were associated with lipid. The buoyant density of the higher molecular weight material was 1.15-1.22 g/ml., and that of lower molecular weight material was 1.2-1.33 g/ml. No latency of the enzyme was revealed by freezing and thawing or treatment with detergents. The nature of the enzyme is discussed. It is likely to be a proteinase possibly bound to some kind of membrane fragment.

Adult↗
PubMed1976

Isolation and characterization of human plasma alpha 1-proteinase inhibitor and a conformational study of its interaction with proteinases.

1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.

Amino Acids↗
PubMedDate not supplied

Tumour necrosis factor alpha stimulates resorption and inhibits synthesis of proteoglycan in cartilage.

During inflammatory reactions, activated leukocytes are thought to produce a variety of small proteins (cytokines) that influence the behaviour of other cells (including other leukocytes). Of these substances, which include the interleukins, interferons and tumour necrosis factors (TNFs), interleukin-1 (IL-1) has been considered potentially a most important inflammatory mediator because of its wide range of effects. In vivo it is pyrogenic and promotes the acute phase response; in vitro it activates lymphocytes and stimulates resorption of cartilage and bone. Cartilage resorption is a major feature of inflammatory diseases such as rheumatoid arthritis, and IL-1 is the only cytokine hitherto known to promote it. TNFs are characterized by their effects on tumours and cytotoxicity to transformed cells, but share some actions with IL-1. I report here that recombinant human TNF alpha stimulates resorption and inhibits synthesis of proteoglycan in explants of cartilage. Its action is similar to and additive with IL-1, and it is a second macrophage-derived cytokine whose production in rheumatoid arthritis, or inflammation generally, could contribute to tissue destruction.

Animals↗
PubMedDate not supplied

Identification of a common class of high affinity receptors for both types of porcine interleukin-1 on connective tissue cells.

Interleukin-1 (IL-1) is the name given to the polypeptides produced by activated mononuclear phagocytes which were originally defined as lymphocyte activating factors (LAF). Administration of IL-1 in vivo causes fever and synthesis of acute phase proteins. In vitro they have been shown to cause cartilage and bone resorption, and to stimulate fibroblasts and chondrocytes to make prostaglandins and latent collagenase. IL-1 has therefore been proposed to be an important inflammatory mediator and may be involved in the destruction of cartilage and bone that is a feature of rheumatoid arthritis and other inflammatory diseases of joints. We therefore looked for IL-1 receptors on connective tissue cells which might be targets for therapeutic intervention. Here we report the iodination, to high specific activity and with retention of full biological potency, of the two types of natural porcine IL-1. These ligands have been used to demonstrate high affinity dissociation constant (approximately 10(-10) M) specific binding sites on pig chondrocytes and synovial fibroblasts, human dermal fibroblasts and murine osteoblasts (3,000-5,000 sites per cell). Most interestingly, the two different Il-1 proteins show a similar affinity for a common class of receptors.

Animals↗