Studies on the origin of the glycoproteins of the rabbit vitreous body using a protein-synthesis inhibitor and radioactive fucose and amino acids.
Rabbits were injected intravitreally with [3H]-fucose and were killed at 0.5, 4, 24, and 40 h postinjection. The radiometric measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus fluorography performed on the vitreous bodies of these rabbits were compared with those carried out on excised vitreous bodies of noninjected rabbits incubated with [3H]-fucose for the same periods. The results of these in vitro experiments demonstrated that at all test intervals there were only two lightly labeled bands in the fluorograms, and this was interpreted as nonenzymatic binding of [3H]-fucose to proteins (glycation), occurring in the extracellular environment. In contrast, intravitreal injections resulted in about 14 conspicuously labeled bands, mainly after the longer time intervals, and the nature of these bands was revealed by additional experiments. In another series of experiments, rabbits were injected intravitreally with either [3H]-fucose or [3H]-amino acids and their vitreous bodies were processed as described above. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always higher than that of the proteins labeled with any of the amino acids. In fluorograms, eight bands unequivocally labeled with the [3H]-amino acids were detected, and corresponding bands almost invariably appeared in the [3H]-fucose lane. Cycloheximide, a protein-synthesis inhibitor, blocked almost completely the incorporation of both [3H]-proline and [3H]-fucose into the vitreous proteins as revealed by the radiometric measurements and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)
Haddad
A
A
Departamento de Morfologia, Faculdade de Medicina de Ribeirão Preto, SP, Brasil.
Laicine
E M
EM
D000596:Amino Acids; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism; D005643:Fucose; D006023:Glycoproteins / Q000096:biosynthesis*; D008297:Male; D011817:Rabbits; D014316:Tritium; D014822:Vitreous Body / Q000378:metabolism*
D000596:Amino Acids; D005136:Eye Proteins; D006023:Glycoproteins; D014316:Tritium; D005643:Fucose; D003513:Cycloheximide