Role for the adenosine triphosphate-dependent proteolytic pathway in reticulocyte maturation.

As reticulocytes mature into erythrocytes, organelles and many enzymes are lost. Protein degradation during reticulocyte maturation was measured by monitoring the release of tyrosine from cell proteins. Proteolysis in rabbit red blood cells was directly proportional to the number of reticulocytes and was low in erythrocytes. This process was inhibited by blockers of cellular adenosine triphosphate production and by agents, such as o-phenanthroline, N-ethylmaleimide, and hemin, which inhibit the soluble adenosine triphosphate-dependent proteolytic system. The breakdown of endogenous proteins in reticulocyte extracts was also inhibited by these agents and required adenosine triphosphate. Inhibitors of lysosomal function, however, did not affect proteolysis. Thus, the proteolytic system that degrades abnormal proteins also catalyzes the elimination of proteins during red cell development.

Oxidized proteins in erythrocytes are rapidly degraded by the adenosine triphosphate-dependent proteolytic system.

The rate of protein degradation in rabbit erythrocytes in normally very low. However, when cells were exposed to agents that oxidize cell proteins (nitrite or phenylhydrazine), the degradation of erythrocyte proteins to amino acids increased 7- to 33-fold. This effect was inhibited by the reducing agent methylene blue. Stimulation of proteolysis also occurred in cell extracts and resulted from the production of substrates (damaged proteins) rather than from activation of proteases. Inhibitors of glycolysis and of the soluble adenosine triphosphate-dependent proteolytic pathway decreased the protein degradation induced by nitrite, whereas inhibitors of lysosomal proteolysis had no effect. Thus, the adenosine triphosphate-dependent proteolytic system is present in mature red cells where it may help protect against the accumulation of proteins damaged by oxidation or other means.

Reversion from deficiency of galactose-1-phosphate uridylytransferase (GALT) in an SV40-transformed human fibroblast line.

Control SV40-transformed human fibroblasts can be readily adapted to growth on medium containing galactose as sole hexose source (galactose-MEH). However, most cells from a line of SV40-transformed skin fibroblasts from a patient with galactosemia (galactose-1-phosphate uridylyltransferase (GALT) deficiency) died in galactose-MEM. Surviving cells of this line either grew in completely sugar-free media or had acquired significant amounts of GALT activity. Two presumptive revertant cell lines with GALT activity were characterized in detail. The expression of GALT in these two lines was stable in nonselective conditions. Each had different reaction maximum velocities with respect to uridine diphosphoglucose (UDPg) concentration as compared to residual activity in the parental cell strain or control cells. Both appeared to demonstrate heat-inactivation profiles for GALT than differed from the parental cells or controls. UDPG concentration was found to significantly alter the thermostability of GALT. A competitive radioimmunoassay for GALT showed that these two lines had amounts of the GALT protein comparable to that of the parental cell strain or control cells. The electrophoretic mobility of GALT from the two presumptive revertants was found to differ from control cells. It was concluded that structural gene changes were probably responsible for the apparent reversion in these lines.