Gelatin: a poor substrate for a mammalian collegenase.

A rabbit tumor collagenase was purified more than 5000-fold. In this form it degrades native collagen in helical conformation at 37 degrees C, pH 7.6, into two fragments, but it had little capacity to cleave gelatin, an indication of the importance of higher-order structure of substrate for this enzyme in pure form. It is likely that, in vivo, enzymes other than collagenase degrade gelatin polypeptides produced by primary collagenolysis.

Localization of lysyl oxidase in hen oviduct: implications in egg shell membrane formation and composition.

Lysyl oxidase activity was found in the isthmus (the membrane-forming region) of the hen's oviduct in a copper-rich region proximal to the shell gland. Desmosine and isodesmosine, cross-linking compounds associated with mature elastin, were found in hydrolysates of the shell membrane, confirming the necessity for lysyl oxidase in its biosynthesis. Shell membranes from hens fed a copper-deficient diet or a diet supplemented with beta-aminopropionitrile had a reduced content of desmosine and isodesmosine.

Collagen-like fragments: excretion in urine of patients with Paget's disease of bone.

Patients with Paget's disease of bone excrete, in the urine, polypeptides that have amino acid composition and other properties resembling those of fragments of collagen. The pattern of isotope incorporation in vivo suggests that these fragments are derived from collagen that has been synthesized and rapidly degraded, or that they are rapidly synthesized but not incorporated into tropocollagen molecules.

Collagenase immunolocalization in cultures of rheumatoid synovial cells.

Cultures of rheumatoid synovial cells that have been enzymatically dissociated and are adherent to a culture vessel are morphologically heterogeneous. When these cells are cultured on a collagenous substrate for 2 to 6 days at 37 degrees C in serum-free medium, they produce collagenase. A monospecific antibody to human collagenase has localized the enzyme extracellularly around cytoplasmic extensions of dendritic cells and intracellularly within a few macrophage-like and fibroblast-like cells.

A collagenolytic system produced by primary cultures of rheumatoid nodule tissue.

A collagenase and a neutral protease have been insolated and characterized from primary cultures obtained from rheumatoid subcutaneous nodules. Release of both active enzymes was maximal between the 3rd and 7th days of culture and was stimulated by the presence of small amounts of colchicine (0.1 mug/ml) added to the culture medium. Both the protease and the collagenase from nodule tissue were active at physiologic pH and were inhibited by chelating agents, sulfhydryl compounds, and 1:40 dilutions of human serum. Both enzymes appeared to have a molcular size equivalent to similar enzymes found in cultures of rheumatoid synovium. The nodule collagenase was purified by chromatography on molecular sieve columns followed by affinity chromatography. The pure enzyme cleaved collagen in solution at 24 degrees C at the locus common for mammalian collagenases to act: three quarters of the distance from the amino-terminus. Under the same conditions the purified enzyme cleaved gelatin (denatured collagen) at the same locus. It is likely therefore that the collagenase in rheumatoid connective tissues functions to produce the initial cleavage of collagen and that after the initial reaction products are denatured, proteases digest them into smaller polypeptides more rapidly than does the collagenase itself. Since rheumatoid nodules grow centrifugally at the expense of the palisading fibroblast layer it seems possible that the central necrotic areas are caused by release of collagenase and protease from the highly cellular palisading zone resulting in the destruction of the extracellular collagen matrix.

Collagenases in human synovial fluid.

An enzyme which degrades native collagen at neutral pH has been isolated from cultures of rheumatoid synovium in vitro, but little or no collagenolytic activity has been found in homogenates of fresh rheumatoid synovium. Similar to most other mammalian collagenases this synovial enzyme is readily inhibited by serum proteins. Proteins of synovial fluid are derived largely from serum and synovial fluid from noninflamed joints was found to inhibit synovial collagenase; the inhibitor was destroyed by trypsin, but not by hyaluronidase. Inhibitory activity was reduced in approximately one-half of the fluids from patients with rheumatoid arthritis. In a total of nine synovial fluids, collagenolytic activity was detectable. This activity was not present in constant amounts in synovial fluids aspirated at different times from the same patient and tended to vary inversely with the titer of inhibitory proteins. The collagenolytic activity in the synovial fluids from different patients was variably inhibited by serum proteins. Two distinct collagenases were detected in some rheumatoid synovial fluids and separated by gel filtration. One, labeled "B" enzyme, with an estimated molecular weight 20,000-25,000 resembled the collagenase obtained from synovial cultures. The other, labeled "A" enzyme degraded collagen fibrils as well as collagen in solution. Disc electrophoresis on acrylamide gels and electron microscopy of segment long spacing (SLS) aggregates of reaction products of the enzymes at 27 degrees C demonstrated that both "A" and "B" enzymes cleaved collagen molecules at a point three-quarters from the amino terminal end of the molecule. Thus collagen degradation in rheumatoid arthritis could result from the operation of these two collagenases.

Urinary polypeptides related to collagen synthesis.

Of the total urinary hydroxyproline in normal subjects and those with skeletal disorders, between 4 and 20% was nondialyzable. In some patients with Paget's disease of bone, hyperparathyroidism with osteitis fibrosa, hyperphosphatasia, and extensive fibrous dysplasia the total urinary hydroxyproline was sufficiently high to permit purification of this polypeptide hydroxyproline by gel filtration and ion exchange chromatography. The partially purified polypeptides had molecular weights between 4500 and 10,000 and amino acid compositions and physical properties resembling those of gelatin. The polypeptide fractions also contained neutral sugar and glucosamine. These fragments had been shown to be susceptible to cleavage by purified bacterial collagenase suggesting the presence of the sequence-Pro-X-Gly-Pro-Y-. After administration of proline-(14)C to patients with Paget's disease hydroxyproline-(14)C was excreted in the urine. The hydroxyproline-(14)C specific activity reached a peak in 2-4 hr and declined rapidly. The specific activity of the polypeptide (retentate) portion was severalfold greater than that of the raw urine and diffusate. When the labeled urines were subjected to gel filtration the hydroxyproline-(14)C fractions of highest molecular weight which were eluted first from the columns had the highest specific activities. Exposure of the hydroxyproline-(14)C-containing polypeptides to bacterial collagenase rendered them dialyzable. Four patients with hyperparathyroidism and osteitis fibrosa were studied before and after removal of a parathyroid adenoma, a period of transition from a predominance of bone collagen resorption to one of relatively increased bone collagen synthesis. The total urinary hydroxyproline fell rapidly after operation whereas the ratio of the polypeptide fraction to the total rose three- to fourfold. The results of these studies suggest that the urinary polypeptides represent fragments of collagen related to collagen synthesis. Changes in the ratio of these peptides to total hydroxyproline in the urine may serve as an index of new bone formation in patients with skeletal disorders.

Activation in vitro of rheumatoid synovial collagenase from cell cultures.

Rheumatoid synovial cells dissociated from matrix and adherent to culture dishes released a latent form of collagenase into culture medium. Previous studies have shown that the latent enzyme does not complex with alpha2-macroglobulin and binds to fibrillar substrate. We now show that serum-free culture medium of the synovial cells contains an inhibitor of collagenase as well as latent enzyme; the two were separated on a column of acrylamide/agarose. Latent collagenase (estimated mol wt 45,000-49,000) was transformed by trypsin to active collagenase of approximately equal to mol wt 33,000. When mixed with inhibitor the active enzyme formed an inactive complex again with approximately equal to mol wt 45,000-49,000. The inhibitor(s) itself was found in one major peak of mol wt 33,000-35,000 and several minor peaks eluting with lower apparent molecular weight. Mersalyl, an organic mercurial compound, effectively activated latent collagenase producing an active enzyme with approximately equal to mol wt 33,000. Bacterial collagenase did not activate latent enzyme. We suggest that latent rheumatoid synovial collagenase, as it is harvested from synovial cells in culture, is an enzyme-inhibitor complex.

Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.

Characterization of a particulate pathway for copper in K562 cells.

More than half of the 67Cu recovered from K562 cells following a brief incubation with 67Cu-ceruloplasmin was recovered in particulate fractions of the cell. The fractions in Percoll had densities that ranged between 1.040 and 1.060 g/dl. In as early as 5 min, two fractions, densities of 1.051 and 1.056, respectively, were discernible. Components in the 1.051 fraction tested positive for clathrin and catalase. Those in the 1.056 fraction sedimented near the marker for lysosomes. The 67Cu in both fractions was stable to treatment by EDTA, nitrilotriacetate, alpha,alpha'-dipyridyl, heparinase, and ascorbate, but dissociated when treated with pronase, trypsin, or sodium dodecylsulfate. Continuous incubation with 67Cu-ceruloplasmin intensified the 67Cu activity in the 1.051 and 1.056 fractions. Cells incubated with 125I-transferrin displayed the label primarily in the 1.051 fraction. Continuous incubation intensified the label but unlike 67Cu, it did not shift to lighter or heavier fractions. Electron micrographs of the 1.051 fraction showed fields dominated by membranous structures some of which were enclosed. Micrographs of whole cells showed numerous invaginations resembling coated pits with sealed structures along and beneath the membrane surface suggesting the membrane was engaged in a rather extensive endocytosis. These data provide evidence that a large fraction of Cu from ceruloplasmin enters the K562 cell bound to membranous-like vesicles, part of which are sealed and coated with clathrin. This particulate pathway accounts for most of the copper entering the cell.

A role for ascorbic acid in copper transport.

Scurvy-like symptoms have been seen in experimental copper deficiency. This forecasts a role for the vitamin in copper metabolism. Ascorbate has been known to antagonize the intestinal absorption of copper. More recent studies have characterized a postabsorption role for ascorbate in the transfer of copper ions into cells. The vitamin reacts directly or indirectly with ceruloplasmin, a serum copper protein, specifically labilizing the bound copper atoms and facilitating their cross-membrane transport. Ascorbate at physiological levels and above impedes the intracellular binding of copper to Cu,Zn superoxide dismutase. The mechanism is unclear but nonetheless suggests both positive and negative regulatory functions for ascorbate in copper metabolism.

Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas. Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase. Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents. BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment. Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome. Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form. With transcript expression, the cells showed a capacity to release copper into the medium. BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene. BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper. We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism. The vesicle model may help us understand the transport of copper across the placenta and all cells in general.

Pathogenesis of rheumatoid arthritis.

Many types of cells are activated and transformed in rheumatoid synovium, thereby contributing to amplification of the disease process. The immune response in rheumatoid arthritis is probably initiated by an antigen, although there is some evidence that anticollagen antibodies develop in response to tissue destruction, after rheumatoid arthritis has evolved clinically. Early inflammation in the synovium is characterized by a striking vascular proliferation, occurring in response to angiogenesis factors released by activated macrophages. Generalized activation of macrophages and lymphocytes typical of the immune reaction in the synovium generates antibody production, including production of rheumatoid factor. Data suggest that immune complexes deposited within cartilage attract polymorphonuclear leukocytes, which then release enzymes onto the cartilage surface. Many products of inflammation act as mediators, driving proliferation of synovial cells. Stellate cells, macrophages, and fibroblasts have been found along the pannus/cartilage junction; by various interactions, these contribute to destruction of cartilage and bone.

Evaluation of pathophysiology and drug effects on rheumatoid arthritis.

The treatment of rheumatoid arthritis has been divided into symptomatic or anti-inflammatory drug therapy, and disease-modifying or remission-inducing drug therapy. A more useful classification may be one that correlates the therapy with the component of the disease process that it affects. In general, nonsteroidal drugs do not affect cells as much as they do some of the mediators that cells produce. The effects of gold and D-penicillamine, although used later in the disease because of their toxicity, probably act early in the cascade of development of the disease by affecting the macrophages and lymphocytes. Prednisone may be effective in inhibiting synthesis by macrophages and fibroblasts of proteinases as well as arachidonic acid metabolites. Retinoids may be prototypes of drugs that selectively inhibit collagenase/proteinase production but do not affect production of normal connective tissue components.

Proliferative diseases.

A unifying concept that excessive proliferation of cells and turnover of cellular matrix contribute significantly to the pathogenesis of several diseases, including cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma and cirrhosis of the liver, is presented. As corollaries to this concept, the following topics are considered: (1) the role of polypeptide hormones and hormone-like mediators in the initiation, promotion and maintenance of proliferative responses; (2) alterations in collagen metabolism and collagenase activity; (3) the role of proteinases; (4) the potential use of inhibitors of proteinases for prevention of disease; and (5) the potential use of inhibitors of proliferative polypeptide hormones for prevention of disease. As specific proteolytic and proliferative biochemical mechanisms which contribute to the pathogenesis of disease become identified, there is a unique opportunity to develop new pharmacologic methods of prevention.

Evaluation of (+)-catechin action on lysyl oxidase activity in aortic tissue.

Small amounts of (+)-catechin (5 mg/kg body wt) administered intramuscularly to 8-day-old chicks raised the lysyl oxidase activity in aorta about 20%. (+)-Catechin had no effect on chicks that were copper-deficient. In the deficient chicks, (+)-catechin treatment prompted a substantially stronger increase in lysyl oxidase activity in response to CuSO4. The observed increments in lysyl oxidase activity in vivo were sensitive to inhibition by beta-aminopropionitrile (BAPN), suggesting that (+)-catechin was affecting the enzyme. (+)-Catechin in the assay medium partially inhibited lysyl oxidase activity. With excess copper ions present, (+)-catechin catalyzed a very strong release of volatile tritium from the substrate proteins. The release of tritium, indicative of lysyl oxidase activity, was not blocked by BAPN, suggesting that the activity in vitro was not enzyme catalyzed.

A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat.

We previously reported that copper efflux from C6 rat glioma cells was blocked by a brief exposure to sulfhydryl reagents p-chloromercuribenzoate (PCMB) and iodoacetamide as well as dicyclohexylcarbodiimide, suggesting the possible involvement of a Cu-transporting ATPase in the efflux mechanism. In this report, we show that copper efflux from PC12 cells, a neuron-like cell line established from rat adrenal pheochromocytoma, is also inhibited by PCMB exposure. Furthermore, we show that both C6 and PC12 cells express a homolog of the Menkes gene (MNK) as detected by RT-PCR with primers designed from a mouse cDNA and confirmed by sequence analysis of the amplified product. An expected 760-bp fragment representing the transduction and phosphorylation domains and a 925-bp fragment encoding the heavy metal-binding domain of Atp7a were amplified from a RNA extract of C6 and PC12 cells. Sequence data revealed that 690 bp of the 760-bp fragment from C6 cells were an identical match to a similar fragment from PC12 cells. Both fragments encoded a 229 amino-acid polypeptide that had a 98.7% sequence homology to mouse Atp7a. In addition, 880 bp from the 925-bp fragment of the two cell lines were identical and encoded a 293 amino-acid polypeptide with 94.5% sequence homology to mouse Atp7a. These data establish that a Menkes-type Cu-transporting ATPase is expressed in rat C6 and PC12 cells and strongly support the hypothesis that both neurons and glia are involved in maintaining Cu homeostasis in the central nervous system.

Iron-copper interactions: some new revelations.

Iron and copper show an uncommon but largely unexplained interdependence, the basis for which is being clarified in hemoglobin-synthesizing cells, yeast, and photosynthetic microorganisms. The interactions of the two metals with membrane transport systems and genetic regulators are more apt to be cooperative than antagonistic.

The pyrroloquinoline quinone (PQQ) coenzymes: a case of mistaken identity.

Recent evidence has failed to support the claim for a pyrroloquinoline quinone (PQQ) cofactor in mammalian enzymes previously reported to have PQQ. The validity of the original analysis now has been questioned, and a second cofactor, topa quinone, has been identified in at least one enzyme.

Menkes' disease: perspective and update on a fatal copper disorder.

Although the causes of the abnormal copper utilization seen in Menkes' disease remain unknown, a candidate gene reported by three laboratories has narrowed the search for the defective or missing factor. These genetic studies also suggest that a copper ATPase may be important in normal copper metabolism.