Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha'-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.
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PMID:The route of secretion of procollagen. The influence of alphaalpha'-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells. 0 39

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of (Mg2+ + Na+ + K+)-ATPase than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for (Mg2+ + Na+ +k+)-ATPase and 5'-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome c reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADPH cytochrome c reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome c reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.
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PMID:Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes. 12 89

Submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation with ethylenediaminetetraacetic acid, and mechanical force. The isolated cells were purified by centrifugation in a Ficoli solution and were maintained in culture for 36 hours. On the basis of trypan blue exclusions, about 70 per cent of the dissociated cells were viable. Electron microscopic observations indicated that the isolated acinar cells and intercalated and striated duct cells retained their essential in situ ultrastructural characteristics. During a 36-hour culture period the number of viable cells declined to about 40 per cent, and the various cell types formed mixed aggregates. The ultrastructural features of the intercalated and duct cells changed relatively little, but the acinar cells revealed several structural alterations. These included a decrease in the number of the secretory granules, fusions of the secretory granules, and an increase in the rough surfaced endoplasmic reticulum. In general, the polarity of acinar cells became less distinct. The endogenous peroxidase activity in the acinar cells gradually diminished during the culture. Isoproterenol when added to the cultured cells failed to stimulate the incorporation of radioactive thymidine or the discharge of the secretory material from the acinar cells.
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PMID:Short term culture of dissociated rat submandibular gland cells. 16 89

Protein synthesis has been studied in a cell-free system from chick embryo, in the presence of homologous RNA isolated from free and endoplasmic reticulum-bound polyribosomes. The two RNA fractions showed equal activities in total protein synthesis. However, while the RNA from bound polyribosomes mainly supported synthesis of high molecular weight, TCA-insoluble polypeptides, the RNA from free polyribosomes was more active in the synthesis of low molecular weight, TCA-soluble polypeptides. Optimal conditions for translation of the two RNA's under study were different when studied in a cell-free system with reduced content of endogenous matrix. Collagen synthesized in the system was identified by collagenase digestion. Collagen synthesis was demonstrated only in the presence of RNA from endoplasmic reticulum-bound polyribosomes, and represented 16-19% of total protein synthesis.
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PMID:Protein biosynthesis in a homologous, cell-free system in the presence of chick embryo RNA isolated from free and membrane-bound polyribosomes. 16 98

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth-walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.
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PMID:Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions. 16 28

The alteration of the structural organization of dermal connective tissue was studied by light and electron microscopy and by biochemical techniques in normal human and in diabetic patients using skin biopsies. Part of the tissue was used for light and electron microscopy, the rest was incubated in the presence of 3H-lysine for four hours. The 3H-lysine labelled biopsies were submitted to a sequential extraction procedure in order to obtain representative macromolecular fractions containing the matrix macromolecules. The extracts were analyzed for their chemical composition and radioactivity. Electron microscopy revealed ultrastructural modifications of the fibroblasts, of the collagen and elastic fibers in the diabetic dermis. Fibroblasts contained an increased amount of electron dense deposits in the cytoplasm and dilated endoplasmic reticulum. The collagen bundles were dissociated. Elastic fibers under the epithelial basal laminae were fragmented or absent. The incorporation pattern of 3H-lysine into these macromolecular fractions was different in the normal and diabetic skin biopsies. The percentage of total radioactivity incorporated increased significantly in the 1M CaCl2 extractable fraction an in the 6M urea extractable fraction and decreased significantly in the collagenase and elastase extracts in diabetic skin biopsy. These results demonstrate the existence of morphological and biochemical alterations in diabetic connective tissue (dermis) reflecting alterations in the relative rates of synthesis and/or degradation of the intercellular matrix macromolecules as well as of their microarchitectural arrangement.
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PMID:[Structural and biochemical alterations of human diabetic dermis studied by H-lysine incorporation and microscopy]. 18 19

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.
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PMID:The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule. 19 95

To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spematids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.
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PMID:Characterization of Sertoli cell-germ cell junctional specializations in dissociated testicular cells. 21 84

Cells that possess the morphology and collagen synthetic capacity of fibroblasts were recovered by bronchofiberscopic subsegmental pulmonary lavage from patients with pulmonary fibrosis, from patients with miscellaneous nonfibrotic lung diseases and from healthy volunteers. Lavage cells were placed in tissue culture, observed for 2 to 6 weeks, and compared with human lavage pulmonary alveolar macrophages (PAM), WI-38 and IMR-90 human fetal lung fibroblasts, and adult lung tissue fibroblasts (CLAC-76). Lavage fibroblsts (LF) were identified as proliferating clones in monolayers of nonproliferating PAM and could be subcultured repeatedly. Fibroglasts were propagated from 28 of the 92 lavage specimens cultured. Time-lapse cinematography showed similar distributions of interdivision times for LF, CLAC-76 and WI-38, but the LF and CLAC-76 lines had slower mean migration rates than the fetal line. Light, scanning, and transmission electron microscopy of LF showed attenuated spindle-shaped cells with interdigitating filopodia, flat surfaces with few microvilli, and containing numerous cytoplasmic polyribosomes and rough endoplasmic reticulum. Extracellular fibrils with the appearance of collagen were seen. Collagen synthesis by LF was measured as 3.9% to 4.9% of the cell-associated protein sensitive to bacterial collagenase. This protein was rich in hydroxyproline, and had an electrophoretic migration pattern identical to known collagen. LF did not contain lysozyme although this enzyme was abundant in fresh and 1-week cultured PAM. Thus LF were similar to human fetal and adult lung tissue fibroblasts in their morphology, tissue culture characteristics, constitutive enzymes and collagen synthetic properties but were distinctly different from PAM.
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PMID:Isolation and characterization of fibroblasts obtained by pulmonary lavage of human subjects. 51 Dec 8

Small (60-90 micrometer) and large (100-130 micrometer) preantral follicles were isolated from adult mouse ovaries by a collagenase-dissection technique. These follicles were composed of resting oocytes surrounded either by granulosa cells, only, or by granulosa and undifferentiated theca cells. Further enzymatic dissociation of primary follicles yielded monodisperse cells characterized by abundant rough endoplasmic reticulum, microfilament-rich pseudopodia and only scant lipid droplets. These cells reaggregated, when explanted in stationary culture, forming epithelial cords and structures macroscopically reminiscent of native ovarian follicles. Anticipated association of follicular cells in epithelial-like monolayers was rare (less than or equal to 10% of all cultured cells). Formation and growth of both follicle-like (FLS) and cord-like (CLS) structures occurred within 24 hours of culture, continued for 14 days, and was inhibited by cytochalasin B, but not by neuraminidase. FLS and CLS, as well as cell monolayers, underwent luteinization, as indicated by the presence in the culture medium of radioimmunoassayable progesterone and by frequent cytological features suggestive of active steroidogenesis. The present report indicates that (a) specific cell affinities exist among immature follicular cells which may play a role in folliculogenesis; and (b) follicular cells are endowed, from their early developmental stages with intrinsic steroidogenic capabilities which become phenotypically expressed after escape from the intraovarian environment.
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PMID:Morphogenetic reaggregation and luteinization of mouse preantral follicle cells. 53 92


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