Purification and characterization of an acid metalloproteinase from human articular cartilage

A metalloprotease that digests cartilage proteoglycan optimally at pH 5.3 has been purified (4400-fold) to homogeneity from 20-g samples of human articular cartilage containing about 100 micrograms of enzyme. This enzyme was cleanly separated from a related neutral metalloprotease with an optimum pH of 7.2. The acid metalloprotease displays 40% of its maximum activity at pH 7.2 and so has significant activity at physiological pH. The protease is calcium-dependent and indirect evidence suggests that it may contain zinc at its active center. It occurs largely in a latent form that can be activated by aminophenylmercuric acetate. The apparent Mr of the latent form is 55,000 and of the active form, 35,000. The isoelectric point is at pH 4.9. The protease activity is inhibited by chelators, Z-phenylalanine, ovostatin, and tissue inhibitor of metalloproteinase from human articular cartilage. It differs from metalloproteinases such as enkephalinase and kidney brush-border protease in its failure to be strongly inhibited by phosphoramidon and Zincov. It cleaves the proteoglycan monomer of bovine nasal cartilage to fragments of approximately 140,000 Da. It cleaves the B chain of insulin at Ala14-Leu15 and Tyr16-Leu17. A survey of 26 cartilage extracts indicates this enzyme is elevated to about 3 times the normal level in human osteoarthritic cartilage and that the tissue inhibitor of metalloproteinase is only slightly diminished. Preliminary evidence points to the presence of a similar acid metalloprotease activity in human polymorphonuclear leukocytes.     

The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.     

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryh?nen et al. (L. Ryh?nen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.     

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.     

Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.     

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)_x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)₁₀ oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)_x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)_x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.     

Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.