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Biomedical subjects

J F Woessner

Publications and source records attributed to J F Woessner.

At least 91 records · Page 5Linked to original sources
PubMed1984

Rise in serum levels of PZ-peptidase, an enzyme involved in collagen breakdown, in human pregnancy and labor.

PZ-Peptidase is an enzyme capable of cleaving small peptides that are produced during the metabolic breakdown of collagen. It is conveniently assayed by use of the synthetic peptide 4-phenylazobenzyl-oxycarbonyl-L-prolyl-L-leucyl-glycyl-L-pro lyl-D-arginine. An improved assay is reported which permits measurements of small amounts of this enzyme in 0.1 ml of serum. The enzyme activity in serum was found to rise from 1.3 U/L (1 U = 1 mumol cleaved per minute) in nongravid patients to 4.0 U/L in patients at term, who display no objective or subjective signs of labor. Patients in active labor show a further elevation of activity to 6.5 U/L. These results are believed to reflect changes in collagen metabolism related to remodeling of the uterus and skeleton in pregnancy and to cervical dilatation and placental separation at the time of labor.

Collagen↗
PubMed1984

Two latent metalloproteases of human articular cartilage that digest proteoglycan.

Human articular cartilage contains very low levels of metalloprotease activity; the activity in 1 g of cartilage is approximately equivalent to the activity of 1 microgram of trypsin. Development of a sensitive assay, based on the digestion of radioactive proteoglycan, has made it possible to study protease activity in 1-2-g specimens of cartilage. Cartilage was extracted with Tris buffer in the cold and with Tris buffer containing 10 mM CaCl2 at 60 degrees C. The extracts were passed through Sepharose 6B; two major and two minor metalloprotease activities were detected. A neutral metalloprotease activity, pH optimum 7.4, was found as a latent form of Mr = 56,000. It could be activated with aminophenylmercuric acetate or trypsin with a resultant decrease of Mr to 40,000. An acid metalloprotease, pH optimum 5.3, also occurred as a latent form of Mr = 50,000. Activation converted this to Mr = 35,000. Removal of calcium ions by dialysis reduced the activity of the neutral enzyme by 80-85% and of the acid enzyme by 100%. Both activities were restored by 10 mM Ca2+. Both enzymes were completely inhibited by 1 mM o-phenanthroline in the presence of excess calcium. This inhibition was overcome by 1 mM Zn2+ and, to a lesser extent, by Co2+. These proteases may be important in the metabolism of the cartilage matrix and in its destruction in osteoarthritis.

Calcium↗
PubMed1984

Extracts of human articular cartilage contain an inhibitor of tissue metalloproteinases.

When human articular cartilage is extracted with 2M-guanidinium hydrochloride at pH 7.5, an inhibitor is obtained that blocks the activity of three metalloproteinases, including collagenase. Molecular-sieve chromatography of the inhibitor gives an Mr value for the inhibitor of 28 500. The inhibitor is stable to heat (60 degrees C, 1h) and acid (pH2, 24 degrees C, 10 min). It is destroyed by trypsin and by reduction and alkylation. It is slowly inactivated by aminophenylmercuric acetate. It binds to concanavalin A-Sepharose and is eluted with alpha-D-1-O-methyl glucopyranoside. Complexes of enzyme and inhibitor are not re-activated by aminophenylmercuric acetate and only partially so by high levels of trypsin. These properties indicate that this inhibitor is a member of the TIMP (tissue inhibitor of metalloproteinases) class. Such an inhibitor, previously found in tissue culture and amniotic fluid, is now shown to be directly extractable from tissue.

Alkylation↗
PubMed1984

Neutral proteases capable of proteoglycan digesting activity in osteoarthritic and normal human articular cartilage.

Proteases have been postulated to account for the progressive disappearance of matrix proteoglycans in osteoarthritic (OA) cartilage. The digestion of endogenous proteoglycans by neutral proteases in human OA cartilage homogenates has been measured and compared with that of normal age-matched controls. Cartilage was obtained from 16 patients at the time of knee arthroplasty and from 7 accident victims. Tissue blocks were cut from the tibial plateau; part was used for histologic grading of the severity of OA and part was homogenized for the quantification of neutral metallo- and serine protease activities, based on the release of digested products from endogenous proteoglycans. Total metalloprotease activity (latent plus active forms) was elevated 3- to 10-fold in all diseased cartilage. This elevation was already significant in mild disease, but was greatest in samples of moderate to severe disease. The active form of the enzyme was highest at the center of erosions and decreased in the margins of the plateau. The digestion of proteoglycans, as distinct from their mere release from the tissue, was demonstrated by chromatography on Sepharose-CL2B and by large pore electrophoresis. Serine protease activity on proteoglycans was much lower than that of metalloprotease. The mean activity was highest in mild disease and declined in the severe disease samples, but the difference between these 2 groups and the controls was not statistically significant. The results of this study are consistent with the hypothesis that the neutral metalloproteases of cartilage are involved in the degradation of proteoglycans in osteoarthritis.

Adult↗
PubMed1983

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures.

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300-1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60 degrees C) and resistant to inactivation by trypsin (2 h, 37 degrees C, 10 microgram/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.

Animals↗
PubMed1983

Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin.

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.

Amino Acid Sequence↗
PubMed1983

Collagen, collagenase and collagenolytic activity in rat Graafian follicles during follicular growth and ovulation.

Follicles were dissected from the ovaries of immature rats at intervals after subcutaneous injection of 20 IU of pregnant mare's serum gonadotropin. A surge of luteinizing hormone was observed at 54 h and ovulation occurred at 64-66 h. The follicular volume between 36 and 48 h, then doubled again shortly before ovulation. The collagen content of the follicles increased 3-fold from 35 to 56 h, but decreased significantly (25%) from 61 to 66 h. Follicle homogenates, activated with trypsin or aminophenylmercuric acetate, digested Type I collagen at 28 degrees C to produce typical of a true collagenase. Collagenolytic activity assayed against endogenous collagen at 37 degrees C did not change significantly between 38 and 66 h.

Animals↗
PubMed1983

Collagenase and collagenolytic activity in human osteoarthritic cartilage.

Forty-nine specimens of human cartilage were taken from 3 sites on the tibial plateau (center of osteoarthritic lesion, edge of lesion, and remote site) and graded histologically by the scale of Mankin. The tissue was homogenized and centrifuged to obtain an insoluble pellet. This was resuspended in buffer and incubated at 37 degrees C, pH 7.5. Collagen digestion was quantitated by the release of hydroxyproline-containing peptides. The highest collagenolytic activity (4.6%) was found in the center of lesions, declining in remote sites to 2.4% and in controls to 1.1%. Moderately severe disease of grade 6--9 had the highest collagenolytic activity. Approximately 55% of the metal-dependent collagenolytic activity was in a latent form, activatable by amino-phenylmercuric acetate; the remainder was self-active. A method was developed for the extraction of collagenase from cartilage; the extracted enzyme produced the typical 75:25 cleavage products of type I collagen.

Aged↗
PubMed1983

Collagenolytic activity and collagen matrix breakdown of the articular cartilage in the Pond-Nuki dog model of osteoarthritis.

Recent reports on the Pond-Nuki model of osteoarthritis in the dog have provided evidence for partial disruption of the collagen network. The possibility of collagenase involvement in these localized changes was studied. Animals were killed 2, 4, 8, and 12 weeks after surgery. The left knee served as a sham-operated control. Cartilages from femoral condyles were processed for light and electron microscopy and assayed for collagenolytic activity by a direct tissue assay, based on the measurement of digestion of endogenous cartilage collagen. Animals killed at 2 and 4 weeks showed fibrillation and mild erosion of femoral condyles, which usually progressed to ulceration by 8 and 12 weeks. Electron microscopy demonstrated fiber disruption of the mid-zone perilacunar collagen as early as 2 weeks after the operation. Total collagenolytic activity, measured after activation by aminophenylmercuric acetate, was significantly higher in decreased cartilage than in controls of 2, 4, and 8 weeks; the peak value was at 4 weeks. Collagenase was shown, by its specific action on type I collagen, to be present at 2 and 4 weeks; however, other metalloproteases may also contribute to the digestion. The correlation between increased collagenolytic activity and the early osteoarthritic changes in cartilage suggests a role of this enzyme activity in the disease process.

Animals↗
PubMed1980

The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan.

1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme--inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15--30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7--7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.

Animals↗
PubMed1980

Effect of oestradiol on the post-partum rat uterus: peroxidase activity and collagen breakdown.

Treatment of parturient rats with 100 micrograms oestradiol/day caused a significant retardation of uterine involution and collagen breakdown. The post-partum uterus had a peroxidase activity of 180 mumol H2O2 reduced/min per uterus. Treatment with oestradiol caused an eight- to tenfold increase in this activity within 3--4 days. Treatment of rats with 4 mg cortisol/day commencing 3 days prepartum had no effect on uterine peroxidase acitvity but it blocked the oestradiol-induced increase in peroxidase. Cortisol had no effect on collagen breakdown and did not reverse the inhibition of collagen breakdown produced by oestradiol. It is postulated that the effects of oestradiol on peroxidase activity are mediated by uterine epithelial cells but that oestradiol effects on collagen breakdown may be mediated by another cell type.

Animals↗
PubMed1979

Separation of collagenase and a metal-dependent endopeptidase of rat uterus that hydrolyzes a heptapeptide related to collagen.

1. The synthetic peptide, 2,4-dinitrophenyl-L-Pro-L-Leu-Gly-L-Ile-L-Ala-Gly-L-Arg-amide (DNP-peptide) was tested as a potential substrate for uterine collagenase. Rat uteri were homogenized and the insoluble fraction was extracted at 60 degrees C to obtain collagenase. The extracts were chromatographed on Sephadex G-150 to yield two peaks of DNP-peptide hydrolyzing activity. Peak I was completely inhibited by EDTA and had a molecular weight greater than 100 000. Peak II was inhibited about 90% by EDTA and had an apparent molecular weight of about 70 000. 2. Peak II coincided closely, but not exactly, with the peak of collagenase activity. It differed from collagenase in heat stability, binding properties on CM-Sephadex and failure to display latency. 3. Peak II represents a new endopeptidase activity. It has a pH optimum of 7 and it cleaves the DNP-peptide at the Gly-Ile and, possibly, the Leu-Gly bond. 4. The DNP-peptide is not a satisfactory substrate for the assay of impure collagenase preparations nor does it inhibit the action of collagenase on collagen substrate when added in 30-fold molar excess.

Animals↗
PubMed1979

Total, latent and active collagenase during the course of post-partum involution of the rat uterus. Effect of oestradiol.

1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.

Animals↗
PubMed1977

PZ-peptidase from chick embryos. Purification, properties, and action on collagen peptides.

PZ-peptidase is an endopeptidase that cleaves the synthetic substrate developed for clostridial collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (PZ-peptide). The peptidase has been purified to homogeneity from chicken embryos. The enzyme has a pH optimum of 7.5 to 8.5, and isoelectric point of 5.0, and a molecular weight of 77,000. The kinetic parameters at pH 8 and 37 degrees are: Km = 2 X 10(-4) M and Vmax = 4.2 mumol/min/mg of protein. The enzyme is inhibited by p-hydroxymercuribenzoate (100%), N-ethylmaleimide (60%), and chelating agents (40 to 60%). Maximum activity is attained in the presence of reducing agents and Ca2+, Sr2+, or Mg2+. The peptidase has no detectable action on casein, serum albumin, collagen, collagen alpha chains, various collagen peptides (alpha1)(I)-CB2, alpha1(I)-CB3, alpha1(I)-CB4), (Gly-Pro-Pro)10, or (Gly-Pro-Pro)5. It does catalyze the hydrolysis of the Hyp--Gly bond in the 17-residue collagen peptide alpha1(II)-CB6-C2 and it partially digested a mixture of collagen peptides of molecular weight 350 to 2500. A role of this peptidase in collagen breakdown appears to be restricted to a late stage when degradation products would fall in the range of 5 to 30 residues.

Animals↗
PubMed1977

A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase.

1. The involuting rat uterus displays an extremely rapid breakdown of collagen. Collagenase activity can be assayed directly in the insoluble 6000g pellet of uterine homogenates. At 1 day post partum, about 85% of this collagenase activity is in a latent form. 2. This latent form can be activated by trypsin or by a serine proteinase present in the uterine pellets. 3. The activating enzyme of the tissue is inhibited by a wide spectrum of trypsin inhibitors, including Trasylol, soya-bean and lima-bean trypsin inhibitors, snail inhibitor and di-isopropyl phosphoro-fluoridate. Partial inhibition is produced by benzamidine, phenylmethanesulphonyl fluoride, epsilon-aminohexanoate, leupeptin, antipain and alpha1-antitrypsin. Ovomucoid, 7-amino-1-chloro-3-tosylamido-1-heptan-2-one and 1-chloro-4-phenyl-3-(N-benzyloxy-carbonyl)amino-L-butan-2-one are not inhibitory. 4. Extraction of uterine pellets with 0.1 M-CaCl2 at 60 degrees C releases both latent and active collagenase. Exclusion chromatography on Sephadex G-100 gives an apparent molecular weight of approx. 77000 for the latent form and 66000 for the active form. The latent form is suggested to be a zymogen of collagenase.

Animals↗