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Biomedical subjects

Y Kishida

Publications and source records attributed to Y Kishida.

At least 55 records · Page 3Linked to original sources
PubMed1977

Hydroxylation of (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 by prolyl hydroxylase. Evidence for an asymmetric active site in the enzyme.

Previous studies with 14C-labeled synthetic peptides demonstrated that prolyl hydroxylase, which synthesizes the hydroxyproline in collagen, preferentially hydroxylates the fourth triplet from the NH-terminal end of the peptide (Pro-Pro-Gly)5. In the experiments reported here, the prolyl hydroxylase reaction was investigated further by preparing chemically modified derivatives of (Pro-Pro-Gly)5 and by synthesizing 14C-labeled preparations of (Pro-Pro-Gly)10. Essentially, the same kcat value was found for the hydroxylation of (Pro-Pro-Gly)5, N-acetyl-(Pro-Pro-Gly)5, (Pro-Pro-Gly)5 methyl ester, (Pro-Pro-Gly)10, and for larger polypeptide substrates of the enzyme. It appeared therefore that preferential hydroxylation of specific triplets in peptides of the structure (Pro-Pro-Gly)n cannot be explained by differences in the kinetic constants for individual triplets. Hydroxylation of 14C-labeled preparations of (Pro-Pro-Gly)10 demonstrated that the ninth triplet was preferentially hydroxylated over any other triplet. The results were best explained by the hypothesis that prolyl hydroxylase has an asymmetric active site in which binding subsites are located adjacent to but not symmetrical with the catalytic subsite.

Amino Acid Sequence↗
PubMed1975

Two improved methods for preparing ferritin-protein conjugates for electron microscopy.

Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin-antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.

Animals↗
PubMed1975

Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis.

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin-labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.

Animals↗
PubMed1973

Collagen synthesis: localization of prolyl hydroxylase in tendon cells detected with ferritin-labeled antibodies.

An improved procedure was employed for linking ferritin to antibodies against prolyl hydroxylase, the enzyme that synthesizes the hydroxyproline in collagen. By electron microscopy, the enzyme was then found to be localized in cisternae of the rough endoplasmic reticulum of embryonic tendon cells; this indicates that hydroxylation of proline occurs while newly synthesized polypeptides are fed into the cisternae.

Animals↗