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R Myllylä

Publications and source records attributed to R Myllylä.

106 records · Page 6Linked to original sources
PubMed1979

Studies on enzymes of collagen biosynthesis and the synthesis of hydroxyproline in macrophages and mast cells.

The activities of four intracellular enzymes of collagen biosynthesis were assayed in freshly isolated rat peritoneal macrophages and mast cells and compared with the same enzymes in freshly isolated chick-embryo tendon cells. The macrophages were found to contain activities of all four enzymes, those of prolyl and lysyl hydroxylase being 7 and 12% respectively of those in the tendon cells when expressed per cell or 3 and 4% when expressed per unit of soluble cell protein. The corresponding values for hydroxylysyl galactosyltransferase and galactosylhydroxylysyl glucosyltransferase activities were about 82 and 68% or 32 and 24% respectively. When the macrophages were incubated in suspension with [(14)C]proline, they synthesized a small but significant amount of non-diffusible hydroxy[(14)C]proline. The synthesis per cell was only about 0.1% of that formed by the tendon cells, and its distribution between the cells and the medium also differed from that in the tendon cells. The hydroxy[(14)C]proline synthesized by the macrophages may be present in the Clq subcomponent of the complement, but its amount was too small to allow any characterization of the protein. All four enzyme activities, and in particular the two hydroxylysyl glycosyltransferase activities, seem to be present in macrophages in a large excess compared with the very low rate of synthesis of hydroxy-proline-containing polypeptide chains. The mast cell extract was found to inhibit all four enzyme activities, but even when corrected for this inhibition, prolyl and lysyl hydroxylase activities in the mast cells were less than 0.08% and the two hydroxylysyl glycosyltransferase activities less than 1% of those in the tendon cells. The intracellular enzyme pattern of collagen biosynthesis in the mast cells is thus completely or virtually completely repressed.

Animals↗
PubMed1978

Further characterization of galactosylhydroxylysyl glucosyltransferase from chick embryos. Amino acid composition and acceptor specificity.

A modified purification procedure, consisting of affinity chromatographies on concanavalin A-agarose, collagen-agarose and UDP-glucose-derivative-agarose and one gel filtration, is reported for galactosylhydroxylysyl glucosyltransferase. The enzyme obtained is entirely pure when studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme protein was rich in glutamic acid + glutamine, aspartic acid + asparagine, glycine and alanine. The enzyme catalysed no significant glucose transfer to any of the glycoproteins tested, except for collagens. This included all the glycoproteins that have previously served as glucosyl acceptors for impure enzyme preparations, thus indicating a high degree of specificity of the enzyme for galactosylhydroxylysine. Galactosylsphingosine would act as a glucosyl acceptor, however. This compound has a close structural similarity to galactosylhydroxylysine in that they both have an unsubstituted amino group next to the hydroxy group to which the galactose is attached.

Amino Acids↗
PubMed1977

Isolation of collagen glucosyltransferase as a homogeneous protein from chick embryos.

Collagen glucosyltransferase was isolated as a homogeneous protein from chick embryos by a procedure consisting of ammonium sulphate fractionation, two affinity chromatographies and two gel filtrations. The specific activity of the purified enzyme was 32,000 times that of the 15,000 x g supernatant of the embryo homogenate, and the enzyme was pure when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis using three different gel compositions. The molecular weight of the enzyme was about 72,000-78,000 by sodium dodecyl sulphate polyacrylamide gel electrophoresis, the value being dependent on the gel composition. The apparent molecular weight by gel filtration was dependent on the purity and protein concentration. The sedimentation coefficient S20,w was 4.7. The data suggest that the enzyme molecule consists of one polypeptide chain.

Animals↗
PubMed1977

A new modification of spectrophotometry in the investigation of amniotic fluid by amnioscopy.

A new spectrophotometric application to amnioscopy for the estimation of the colour of amniotic fluid is presented. Three light-emitting diodes (LEDs) with peak wavelengths of 565 nm (green), 581 nm (yellow), and 645 nm (red) act as the light sources, and the light reflected from the amniotic fluid is measured using a photodiode. The greatest advantages of our new application are the short recording time of about 15 s. and the simplified mechanical performance of the measurement, which reduce errors. Preliminary clinical results show that green colouration can be easily discriminated from yellow and clear amniotic fluid. Sources of error and improvements to the method are discussed.

Amniotic Fluid↗
PubMed1976

Studies on the mechanism of collagen glucosyltransferase reaction.

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.

Animals↗
PubMed1976

Affinity chromatography of collagen glycosyltransferases on collagen linked to agarose.

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.

Animals↗
PubMed1976

Collagen glucosyltransferase. Partial purification and characterization of the enzyme from whole chick embryos and chick-embryo cartilage.

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.

Animals↗
PubMed1975

Glucosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate.

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.

Animals↗