Abstract
Protease and diazobenzene sulfonate have been used to probe the transverse topology of the microsomal glucose‐6‐phosphatase system. Treatment of intact microsomes with these reagents under the conditions used here did not affect the permeability of the membrane to mannose 6‐phosphate, nucleoside diphosphatase, or dextran of 70 000 molecular weight. Nor did these treatments inactivate the hydrolytic site of glucose‐6‐phosphatase, a finding in agreement with earlier conclusions that this site is on the inside of the membrane. On the other hand, treatment of intact microsomes with diazobenzene sulfonate or proteases does inactivate (or increase the apparent Km of) some other component which is rate‐limiting for glucose‐6‐phosphatase activity in intact but not in disrupted microsomes. The simplest explanation for this phenomenon is that there is a protein carrier in the microsomal membrane which transports glucose 6‐phosphate from the medium to the lumen, where it is hydrolyzed, and that diazobenzene sulfonate and proteases attack this carrier. The lack of effect of these reagents on microsomal inorganic pyrophosphatase activity suggests that the glucose‐6‐phosphate carrier cannot transport pyrophosphate. Finally, it was found that treatment of microsomes with ammonia breaks down their permeability barrier but also removes significant amounts of microsomal phospholipid and inactivates a number of microsomal enzymes. We do not recommend it as a general approach to altering microsomal permeability.
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