Collagen enzyme complexes

Preparation of Collagen-Enzyme Complexes. Collagen-enzyme complexes were prepared by the membrane impregnation method (9,... 

Enzyme Binding Capacity Determination. Two steps or stages are involved in formation of a stable collagen/enzyme complex. 

The inducers of these alterations range from certain proteolytic enzymes, in particular thrombin, to large molecules such as collagen, immune complexes, zymosan (alpha 41) and cationic polymers (alpha 28), and, finally, low molecular weight compounds such as adenosine-5-diphosphate (ADP), adrenaline, vasopressin, and serotonin. This wide variety of materials, all of them capable of eliciting the same result, suggests that.upon quite different stimuli of the plasma membrane, the same sequence of events is triggered off. These events can be subdivided by several criteria in different phases ... 

Co-expression of the human a- and p-subunits in the yeast Pichea pastoralis produces only trace amounts of active tetramer, with the majority being present in an unassembled form. Co-expression with human type III collagens, however, increases this assembly level tenfold. This indicates that collagen synthesis and the formation of an active prolyl 4-hydroxylase complex are mutually dependent processes (Vuorela et al, 1997). A similar observation has been noted for baculovirus encoded enzymes in insect cells (Lamberg et al, 1996). These findings support the hypothesis that this unusual control mechanism may be a common feature of collagen synthesis in all cell types. 

In contrast to milk, where samples are primarily derived from cows, meat analysis has to be performed in samples of a widely different animal origin including cattle, lamb, swine, poultry, and fish. Muscle is a complex matrix with a pH of 5.7, composed of muscle fibers, various types of connective tissue, adipose tissue, cartilage, and bones. Sarcoplasmic proteins such as myoglobin, and glycolytic enzymes are soluble in water while the myofibrillar proteins such as myosin and actin are soluble in concentrated salt solutions (14). The connective tissue proteins, collagen and elastin, are insoluble in both solvents. 

In in vitro studies penicillamine inhibited angiotensin-con-verting enzyme (ACE) and carboxypeptidase (930). Penicillamine interferes with the functions of the copper-containing enzyme ceruloplasmin, and some of the penicillamine- and copper-containing complexes formed in vivo have a superoxide dismutase effect (931). In patients with scleroderma, penicillamine normalized collagen metabolism, by inhibiting beta-galactosidase activity (932). 

The second mode of crystal formation that occurs in dentin is via matrix vesicles. These are phospholipid delimited packages of specialized enzymes, macromolecular complexes and ions, that induce the precipitation of amorphous calcium phosphate. At some point the latter crystallizes into carbonated apatite crystals, that have no preferred orientation [62], These appear smaller and denser than the crystals that form in the collagen framework. 

Since protein complex formation and Ca2+ are critical to cell fixation within a tissue, dissociation media usually contain some type of proteolytic enzyme and the Ca2+ chelator, EDTA. The proteolytic enzyme can be of general specificity, such as trypsin, or can be a more targeted enzyme, such as a collagenase selective for the collagen-type characteristic of the tissue of interest. Hyaluronidase has been also used with matrix rich in hyaluronic acid, such as for isolation of duodenal entero-cytes. In all cases, the appropriate incubation times and concentrations to achieve cell dispersal, but retain high viability, need to be determined empirically. One factor... 

Progressive fibro-proliferative diseases (e.g. liver cirrhosis, pulmonary fibrosis, rheumatoid arthritis) result in a dramatic increase in collagen synthesis [227], This is preceded by inflammation that correlates with an increased activity of proline and lysine hydroxylase [228], Although they are unlikely to be the primary initiators of these diseases the increased activities of these enzymes may cause other problems. For example, in vitro the enzyme can turn over in the absence of a peptide substrate (but the presence of the 2-oxoglutarate cofactor). In this case stoichiometric amounts of ascorbate are required, probably to reduce the ferryl ion back to ferrous [229]. In vivo, lower concentrations of ascorbate are utilised [229,230], possibly to reactivate the enzyme after a non-productive activation (for example in the presence of a peptide that can bind to the active site, but cannot be hydroxylated). As the amount of proline-hydroxylase activity increases in the fibro-proliferative diseases, the concentration of ascorbate might not be sufficient to reduce these inactive complexes, resulting in the formation of potentially reactive ferryl intermediates. 

The decrease in enzyme sorption by the chemically modified membrane implies that under these experimental conditions, the lysyl E-amino groups function as principle receptor or binding sites for enzyme protein (at least for E. coli g-galactosidase) and that the complexation mechanism involves interaction of the lysyl residues of collagen with enzyme amino acid chains as an initial step in the formation of a stable network of physicochemical bonds. 

As previously discussed, the decrease in enzyme binding capacity of the chemically modified membrane implies that the complexation mechanism involves ionic interactions of lysyl E-amino groups of collagen with enzymic amino acid side chains as a principle step in the formation of a stable network of phyico-chemical bonds. However, part of the effect of carbamylation could be due to a reduction in the average binding constant (K0) of all the sites rather than the complete blocking of active sites. 

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