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Denatured citrate-soluble collagen was coupled to agarose by the cyanogen bromide activation technique, and columns prepared from this material were studied for affinity chromatography of collagen glycosyltransferases. Both collagen glycosyltransferases became bound to the column, the degree of binding and the capacity of the column being higher with the glucosyltransferase than with the galactosyltransferase than with the galactosyltransferase. The addition of Mn2+ enhanced the binding, especially with the glucosyltransferase. The enzymes were eluted from the column with small peptides prepared from collagen, and they were separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase purification of about 5000-fold and a collagen galactosyltransferase purification of about 1000-fold was obtained from chick embryo extract by relatively simple steps.

Monoclonal antibodies against human prolyl 4-hydroxylase (EC 1.14.11.2), an intracellular enzyme of collagen biosynthesis, were produced by fusing spleen cells from BALB/c mice hyperimmunized with human prolyl 4-hydroxylase and mouse myeloma cells (P3/NS 1/1-AG 4-1). Hybridomas from 14 different primary microtiter-plate well cultures produced antibodies to human prolyl 4-hydroxylase; six of them with the highest antibody titer were cloned and antibodies produced by one clone from each of the six lines were further characterized. All of the six cloned hybrids produced antibodies of the IgG class as detected by immunodiffusion. The enzyme antigen used in the present study was a tetramer composed of two pairs of different subunit proteins, alpha and beta. Only one clone which produced antibodies to the alpha subunit was obtained, the other five antibodies being directed against the beta subunit. All the antibodies reacted with the tetramer form of the enzyme. Species cross-reactivity of the antibodies was tested using cultured human, mouse and chick fibroblasts and purified prolyl 4-hydroxylase from chick and mouse sources. None of the antibodies cross-reacted with chick or mouse fibroblasts, as determined by immunofluorescence, whereas one antibody reacted with purified chick and mouse prolyl 4-hydroxylase when examined by the western blotting technique. This antibody caused a strong inhibition of human prolyl 4-hydroxylase activity, but the other five antibodies had negligible inhibitory effect on the activity of the enzyme.

A high correlation was found between the activities of protein disulphide isomerase and prolyl 4-hydroxylase when assayed in cells synthesizing various collagen types or the same type at markedly different rates. The highest activities of both enzymes were found in freshly isolated chick-embryo tendon and cartilage cells, intermediate activities in confluent cultures of human skin and lung fibroblasts and mouse 3T6 fibroblasts, and the lowest values in three human sarcoma cell lines, the difference in protein disulphide isomerase activity between the freshly isolated tendon cells and confluent simian-virus-40-transformed human lung fibroblasts being about 25-fold. All these differences are in good agreement with differences reported between the various cells in their rates of collagen synthesis. A great similarity was also found between the changes in the two enzyme activities measured per cell during the growth of 3T6 fibroblast cultures from the early logarithmic phase to the stationary phase. No correlation was found between protein disulphide isomerase activity and the type of collagen synthesized. The data suggest that protein disulphide isomerase may be involved in the formation of intra-chain and inter-chain disulphide bonds in procollagens, but there is no collagen type-related variation in this enzyme activity of a magnitude that would explain the marked differences in the rates of formation of inter-chain disulphide bonds between the various collagen types.

A purification of over 2000-fold is reported for collagen glucosyltransferase from Triton X-100 extract of whole chick embryos and one of about 160-fold from similar extract of chick embryo cartilage. The addition of the detergent more than doubled the enzyme activity in the homogenates. The purified enzyme preparations from whole chick embryos showed one major band and two or three minor bands in polyacrylamide gel electrophoresis and were entirely free of collagen galactosyltransferase activity. The molecular weight of collagen glucosyltransferase from both sources was about 52000 -- 54000, as determined by gel filtration. In some enzyme preparations an additional form was observed, with an elution position corresponding to a molecular weight of about 130000. Manganese was the most effective metal co-factor for the purified enzyme, but partial replacement could be obtained with Co2+, Mg2+ and Ca2+, whereas no replacement was found with other metals. The activity of the purified enzyme was stimulated by the addition of dithiothreitol to the incubation system and inhibited by preincubation with p-mercuribenzoate. UDP-glucose or the collagen substrate partially protected the enzyme against p-mercuribenzoate inactivation in the presence of Mn2+ but not in its absence. Some protection was also noted with Mn2+ alone.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.

The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparations purified about 2500-5000-fold from extract of homogenate of whole chick embryos. Data obtained in experiments on initial velocity and inhibition kinetics of the reaction were consistent with an ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, UDP-glucose and collagen substrate, the addition of Mn2+ being at thermodynamic equilibrium and the binding site of the UDP-glucose to the enzyme not being the same as that for Mn2+ and collagen substrate. Only one metal co-factor seems to be involved in the reaction. The collagen substrate can probably also react in some conditions with enzyme-Mn2+ and with enzyme-Mn2+-UDP, and the UDP with the free enzyme, but in all these instances dead-end complexes are formed. Evidence is presented for an ordered release of the products in the following order: glucosylated collagen, UDP and Mn2+, in which Mn2+ need not leave the enzyme during each catalytic cycle.