Showing papers by "Joseph W. DePierre published in 1978"
TL;DR: A spectrophotometric method for assaying cytochrome P -450 in microsomes contaminated with large amounts of hemoglobin and methemoglobin has been developed and it is necessary to add 0.25 m m ascorbic acid and 2.5 μ m PES (phenazine ethosulfate) to both cuvettes to balance the CO-hemoglobin peak.
174 citations
165 citations
TL;DR: It was found that treatment of intact microsomes with ammonia breaks down their permeability barrier but also removes significant amounts of microsomal phospholipid and inactivates a number ofmicrosomal enzymes.
Abstract: 1
Protease and diazobenzene sulfonate have been used to probe the transverse topology of the microsomal glucose-6-phosphatase system.
2
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.
3
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.
4
The lack of effect of these reagents on microsomal inorganic pyrophosphatase activity suggests that the glucose-6-phosphate carrier cannot transport pyrophosphate.
5
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.
90 citations
TL;DR: Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats, which indicates that the enzyme molecules are not relocated after their insertion into the membrane.
44 citations
TL;DR: A number of alternative assay procedures for epoxide hydrase are also reported, including procedures where an epoxide of a polycyclic hydrocarbon is used as substrate and the products are separated from remaining substrate by gas chromatography or by high-pressure liquid chromatography.
Abstract: Publisher Summary The purpose of this chapter is to provide information on the radioactive assay of aryl hydrocarbon monooxygenase and epoxide hydrase. Polycyclic hydrocarbons are found in cigarette smoke and in the waste products of our technology and may be a major cause of cancer in human beings. Assay of epoxide hydrase using styrene oxide is very simple; extraction with petroleum ether removes remaining substrate, while the product styrene glycol is retained in the aqueous incubation medium. The first step in the metabolism of these compounds by mammalian tissues is catalyzed by the NADPH-dependent, cytochrome P-450-1inked monooxygenase system localized on the endoplasmic reticulum, and the first product is an epoxide. A number of alternative assay procedures for epoxide hydrase are also reported, including procedures where an epoxide of a polycyclic hydrocarbon is used as substrate and the products are separated from remaining substrate by gas chromatography or by high-pressure liquid chromatography.
34 citations
TL;DR: It is concluded that the benzpyrene monooxygenase system and epoxide hydratase may form a complex of unique stoichiometry in the membrane of microsomes from control rats, but that such a complex is not consistent with the distributions obtained after methylcholanthrene induction.
17 citations