Effect of storage and in vitro ischemia on the ultrasture of microsomal membranes and on microsomal enzymes.
Pieces of liver (in vitro ischemia) and isolated microsomes were subjected to incubation at 4 degrees C and 37 degrees C for various time intervals. The effects on microsomal protein, phospholipids, and cholesterol and on microsomal phosphatases and electron transport enzymes were followed as a functional of time and temperature. NADH-cytochrome c reductase was very labile and was completely inactivated by 1 h, whereas G6Pase lost 50% of its activity after 2 h at 37 degrees C. IDPase and NADPH-cyt. c red. were of intermediate susceptibility whereas cytochromes b5 and P-450 were the most stable enzymes assayed. After 24 h of incubation of isolated microsomes at 37 degrees C there was no significant detachment of membrane components (protein, PLP or cholesterol), indicating that the inactivation of the enzymes was not primarily attributable to their solubilization. Instead, experiments with 14C-leucine and 14C-glycerol prelabeled microsomes demonstrated that the proteins detached from microsomes during incubation originated mainly from the intravesicular space due to repture of the microsomal membranes. The addition of a lysosomal extract during incubation did not alter either the rate of inactivation of the enzymes or the proportion of solubilized membrane components indicating that attack from the outside by proteolytic enzymes is not the mechanism for enzyme inactivation. There was no apparent correlation between the rates of inactivation of enzymes in vitro and their calculated half-lives in vivo or their postulated intramembranous localization. Ultrastructurally, enzyme inactivation was initially associated with alterations of the microsomal membranes, such as vesicle aggregation, membrane rupture, loss of unit membrane structure, and subsequently, thickening of membranes and transformation of the microsomes into nonrecognizable amorphous material.
D000818:Animals; D002784:Cholesterol / Q000032:analysis; D007511:Ischemia / Q000473:pathology*; D008297:Male; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology / Q000648:ultrastructure*; D010743:Phospholipids / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D011506:Proteins / Q000032:analysis; D051381:Rats; D013696:Temperature; D013997:Time Factors
D010743:Phospholipids; D011506:Proteins; D002784:Cholesterol; D010744:Phosphoric Monoester Hydrolases