Short-chain dolichols of defined chain length as cofactors in reactions of the microsomal dolichyl-phosphate cycle and transglycosylations.
1) The biological cofactor and carrier activities of dolichyl phosphates of low isoprene multiplicity (n) and defined geometry, (synthesized according to L. Jaenicke and H.-U. Siegmund, Chem. Phys. Lipids 51 (1989) 159-170), were assayed in different transfer reactions of the microsomal dolichyl-phosphate cycle against natural pig liver dolichyl phosphate (n = 18 to 20). 2) The apparent Michaelis constants and maximal velocities were determined from initial reaction rates for the transfer from GDP-mannose, UDP-N-acetylglucosamine, and UDP-glucose to the synthetic truncated dolichyl phosphates. They afford quantitative comparison and show increasing biological activities from dolichyl-6 phosphate to dolichyl-11 phosphate, which is about as active as the natural mixture. This is in accord with previous findings on the starting reactions of the cycle. 3) Truncated dolichyl diphosphochitobioses, biosynthesized in vitro from synthetic dolichyl phosphates, were used as acceptors for nucleoside diphosphohexoses in solubilized membranes. All of them show about the same activity. The kinetics and yield were determined for each of the transfers. Activity is increased by adding UDP-glucose. The inactive very short-chain dolichol compounds do not interfere with the transfer to active longer chain dolichols. 4) The oligosaccharides produced by transfer of mannose and glucose to truncated dolichyl diphosphate-bound chitobiose were isolated and analysed for sugar multiplicity. The heptasaccharide and the un-decasaccharide are accumulated most, pointing to the transport across the endoplasmic membranes (ER) as the rate limiting reaction. 5) The truncated dolichyl-diphosphate-bound oligosaccharides are transferred to protein(s) by the crude, solubilized microsomal preparation independent of chain length of the cofactor/carrier, yet with increasing yield as shown by enzyme immunoblot analysis.
Berendes
R
R
Institut für Biochemie, Universität zu Köln.
Jaenicke
L
L
D000818:Animals; D002417:Cattle; D004288:Dolichol Phosphates / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006601:Hexoses / Q000378:metabolism; D066298:In Vitro Techniques; D008862:Microsomes, Liver / Q000378:metabolism*; D013379:Substrate Specificity
D004288:Dolichol Phosphates; D006601:Hexoses