Seppo Lindy

Human leukocyte collagenase: Characterization of enzyme kinetics by a new method

Abstract Human collagenase was partially purified from polymorphonuclear leukocytes, and a new method for assay of the collagenase activity was developed. The assay employs native radioactive collagen in soluble form as a substrate. The enzyme incubations are performed at 25°C which is below the melting temperatures of the cleavage products TCA and TCB, and these peptides are quantitatively recovered by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Employing this method, an apparent K m value of 1.04 × 10 −6 m for human leukocyte collagenase using type I collagen as a substrate was measured.

Protocollagen Proline Hydroxylase Activity in Rat Heart During Experimental Cardiac Hypertrophy

Cardiac hypertrophy was produced in rats by constricting the abdominal aorta subdiaphragmatically. The weight of the left ventricle was significantly elevated 2 days after aortic constriction and was 19% higher than in shamoperated animals at 11 days. The activity of protocollagen proline hydroxylase (PPH), an enzyme participating in collagen biosynthesis, and the hydroxyproline content of the heart muscle were determined. PPH activity was increased at 2 days after aortic constriction and declined thereafter, being still above the control level at 11 days. The hydroxyproline content of the left ventricle was significantly increased at 11 days in hypertrophied heart muscle compared to controls. The present results suggest that in cardiac hypertrophy an early connective tissue activation occurs. Later, this leads to connective tissue accumulation. The increased collagen content may give support to the cardiac muscle contracting against systolic overload.

Activation of human leukocyte collagenase by compounds reacting with sulfhydryl groups

Abstract Human collagenase was partially purified from the granules of polymorphonuclear leukocytes by gel filtration, and the effects of two sulfhydryl reagents, N -ethylmaleimide and p -aminophenylmercuric acetate, on the enzyme activity were studied. The enzyme activity was assayed by incubating with soluble [ 14 C]proline-labeled type I collagen, and the rate of collagen cleavage was quantitated by isolating the specific cleavage products by SDS-polyacrylamide gel electrophoresis. Results demonstrated that the collagenase, which was at first mostly in a latent form, was rapidly activated by these two sulfhydryl reagents. The enzyme activity, however, returned gradually to the control level in the presence of the sulfhydryl reagent or after removal of an excess of the reagent. The enzyme activity, after activation with N -ethylmaleimide, could be returned to the control level by the addition of a 2-fold molar excess of cysteine. The heat stability of the enzyme activity before and after activation by N -ethylmaleimide was also tested. The results indicated that the initial enzyme activity, before the activation, was stable at 60°C for at least 5 min, and the enzyme could be subsequently activated by N -ethylmaleimide to the same extent as an unheated control. If, however, the enzyme was first activated by incubating in the presence of N -ethylmaleimide and subsequently incubated at 60°C, a marked decrease in the enzyme activity as a result of the 5 min heating was noted. The results of the present study indicate that human leukocyte collagenase can be activated by compounds reacting with thiol groups.

Collagenase Reserves in Polymorphonuclear Neutrophil Leukocytes from Synovial Fluid and Peripheral Blood of Patients with Rheumatoid Arthritis

Degradation of cartilage in rheumatoid arthritis (RA) may be in part due to release of collagenase from specific granules of polymorphonuclear neutrophil leukocytes (PMNs) during degranulation. We decided to study, not synovial fluid (SF) collagenase, but PMN collagenase reserves. PMN were isolated from parallel SF and peripheral blood (PB) samples obtained from 7-arthritis patients. PMNs were stimulated in vitro by tetradecanoyl-phorbol-13-acetate (TPA). Collagenase activity in the supernatant without and with phenylmercuric chloride activation was studied. Compared to PB PMNs, there was no consistent decrease in the total collagenase reserves in the inflammatory SF PMNs. This suggests that the release of collagenase in the inflammatory synovial fluid does not deplete SF PMNs of the collagenase synthesized at the myelocyte stage. The role of PMN collagenase in pathogenesis of cartilage destruction would then seem to be more dependent on local release and autoactivation at cartilage surface by adherent PMNs and not excessive collagenase release from free floating SF PMNs at single cell level. Furthermore, under the experimental conditions used the proportion of collagenase released in active form was higher in SF PMN specimens than in PB PMN specimens (p less than 0.01). The predominant collagenous component of adult cartilage, native type II collagen, was degraded by PMN collagenase as fast as native type I collagen. These findings suggest an important role for this enzyme in destruction of the free cartilage surface in RA.

Increased protocollagen proline hydroxylase activity in synovial tissue in rheumatoid arthritis

Abstract Protocollagen proline hydroxylase activity was assayed in biopsy specimens of human synovial tissue. The enzyme required ascorbate, α-ketoglutarate, ferrous iron and molecular oxygen for its activity. The activity, determined from synovial tissue of 18 patients with rheumatoid arthritis, was increased as compared with values obtained from control patients.

Denaturation of Lactic Dehydrogenase Isozymes and its Clinical Application

IT is known that lactic dehydrogenase (LDH) can be inactivated by treatment with various protein denaturants, for example, urea and guanidine hydrochloride1,2. In the denaturating process the tertiary structure of the enzyme is destroyed with the concomitant loss of enzymatic activity. There is evidence that fast-migrating isozyme (LDH1), a predominant fraction in heart tissue, is less sensitive to these denaturating processes than the slower migrating isozymes3,4. The various isozymes are known to differ from each other in their amino-acid composition and this may play a central part in susceptibility to denaturation.

Gold sodium thiomalate activates latent human leukocyte collagenase.

Gold sodium thiomalate, a drug used widely in the therapy of rheumatoid arthritis, was found to be an activator of latent human polymorphonuclear leukocyte collagenase. The activation was demonstrated by two distinct and independent collagenase assays: (i) by recording with a spectrophotometer at 227 nm the enzyme‐induced increase in ultraviolet difference absorbance of native type I collagen connected to the cleavage of collagen at 37°C[(1986) Eur. J. Biochem. 156, 1‐4] and (ii) by SDS‐polyacrylamide gel electrophoresis analysis of formation of specific products of collagen resulting from collagenase cleavage at 25°C. Activation of latent collagenase by gold sodium thiomalate appeared to be of the same magnitude as by the known activator phenylmercuric chloride.

Increased collagen prolyl hydroxylase activity in the aortic wall of rabbits exposed to chronic hypoxia

The activity of collagen prolyl hydroxylase in aortic wall was studied in rabbits exposed to chronic 10% ambient oxygen tension for 30 days. Prolyl hydroxylase in rabbit aorta was shown to be similar to the enzyme from other sources in that it required molecular oxygen, alpha-ketoglutarate, ferrous iron and ascorbate for its activity. The activity of prolyl hydroxylase was increased to 180% of controls in the intima-media samples from rabbits exposed to hypoxia. No atherosclerotic lesions could be seen in arteries of animals kept in chronic hypoxia. If the arteries of rabbits were injured with a single mechanical dilatation, the activity of prolyl hydroxylase increased more than 2-fold, as reported previously. The exposure of these animals to chronic hypoxia further elevated the prolyl hydroxylase activity.

Latent human leukocyte collagenase can be activated by gold thioglucose and gold sodium thiomalate, but not by auranofin

Gold thioglucose and gold sodium thiomalate were shown to be potent activators of latent human leukocyte collagenase. No activation by auranofin was noted. The activation may proceed through the action of gold on the essential sulfhydrylgroups of latent enzyme and, thereby, mimick the action of the known organomercurial activators.

Lactate dehydrogenase activity in high pyruvate concentrations modified by urea

Abstract The effect of urea on human LDH1 and LDH5 isoenzymes and on the LDH activities of human heart and liver homogenates was studied at various pyruvate concentrations. It was demonstrated that in high pyruvate concentrations (from 1.5 · 10−3M upward) the activity of LDH1 and heart homogenate was greater in urea than when measured without urea. On the contrary, the activity of LDH5 and liver homogenate was almost completely destroyed in urea. using a high pyruvate concentration (4 · 10−3M) the rather preliminary clinical results show that the serum LDH activity in patients with myocardial infarction is greater in urea than without urea. In liver diseases the serum LDH activity was lower in urea than without urea.