Cytochrome P-450scc induces vesicle aggregation through a secondary interaction at the adrenodoxin binding sites (in competition with protein exchange.

Addition of bovine adrenal cytochrome P-450scc to small unilamellar dioleoylphosphatidylcholine vesicles (DOPC-SUV) produces a complex sequence of interactions, indicating exceptional cytochrome mobility. First, cholesterol transfer from cytochrome to vesicles indicated rapid dissociation of P-450scc oligomers and integration of monomers into the membrane (delta A 390-420 nm; t1/2 = 2 s). After 10-15 s, P-450scc-induced aggregation of the vesicles starts, as indicated by increased turbidity (delta A 448 or 520 nm; complete in 6-8 min). Fluorescence quenching experiments indicate that this aggregation does not lead to measurable vesicle fusion during this period. Aggregation is prevented by mild heat denaturation of P-450scc, by addition of anti-P-450scc IgG, and also by 1:1 complex formation with the electron donor adrenodoxin (ADX). P-450scc, therefore, links two vesicles through two separate domains involved in, respectively, membrane integration (lipophilic) and ADX binding (charged). Although completely bound by DOPC-SUV, as evidenced by Sephadex elution, P-450scc has access within 1 min to cholesterol in secondary SUV. This is indicated by spectral changes (cholesterol complex formation) and by metabolism of secondary vesicle cholesterol. Since cholesterol equilibrates slowly between vesicles (t1/2 = 1-2 h), these changes arise from P-450scc transfer. This transfer was maximally slowed after a 5-min preincubation with primary vesicles, reflecting more extensive integration into the membrane than is necessary for the rapid initial cholesterol transfer to P-450scc. P-450scc transfer probably results from simultaneous interaction of P-450scc with two vesicles that may also initiate aggregation. Weaker integration into primary dimyristoylphosphatidylcholine vesicles facilitates exchange but prevents aggregation. Integration and aggregation are both enhanced by incorporation of 10% phosphatidylinositol into SUV, while exchange is slowed. This mobility of P-450scc is most probably a consequence of the absence of amino-terminal anchoring. P-450scc-induced association of inner mitochondrial membrane segments may contribute to the exceptionally vesiculated structure of adrenal and ovarian mitochondria that parallels increased P-450scc content.

Cholesterol metabolism by purified cytochrome P-450scc is highly stimulated by octyl glucoside and stearic acid exclusively in large unilamellar phospholipid vesicles.

Cholesterol side-chain cleavage (CSCC) catalyzed by purified bovine adrenal mitochondrial cytochrome P-450scc is highly dependent on the vesicles that supply cholesterol. Six-fold higher rates are achieved with large unilamellar dioleoylphosphatidylcholine vesicles (diameter 150 nm) prepared by octyl glucoside (OG) dialysis (DOPC-LUV) than with small sonicated vesicles (diameter 30 nm) (DOPC-SUV) (Vmax = 25 and 4 min-1, respectively. Extensive dialysis that may remove OG decreased Vmax rates for DOPC-LUV almost to rates seen with DOPC-SUV. These dialyzed DOPC-LUV were, however, very sensitive to addition of OG (EC50 = 2.5 microM, 4.3-fold stimulation) while DOPC-SUV were only weakly affected (EC50 = 100 microM, 1.6-fold stimulation). This enhancement of CSCC in LUV by OG only occurred when the cholesterol:DOPC exceeded 0.1 and was associated with a 15-fold increase in the Km for cholesterol. Structural changes in both SUV and LUV at high cholesterol:DOPC ratios (0.1-1) were indicated by decreases in internal volume that were insensitive to OG and did not affect the external diameters. Stearic acid produced a similar stimulation of CSCC in LUV (EC50 = 50 microM) and had no effect on SUV. The Vmax for CSCC, produced by OG activation of DOPC-LUV, is comparable to the highest attained for cytochrome P-450scc (Tween 20/cholesterol). In LUV, a minor proportion of OG (1-5% of cholesterol) is thus sufficient to generate a domain of reactive cholesterol that maintains a near-optimum turnover. This increased CSCC was paralleled by increased binding of cholesterol to P-450scc, suggesting that this cholesterol is more readily donated by the membrane to the cytochrome.

ACTH control of cholesterol side-chain cleavage at adrenal mitochondrial cytochrome P-450scc. Regulation of intramitochondrial cholesterol transfer.

Rat adrenal mitochondria exhibit a linear 2-fold accumulation of cholesterol for 20 min following either in vivo ether stress or ACTH administration, providing cholesterol metabolism is inhibited by aminoglutethimide (AMG). Additional cycloheximide (CX) pretreatment only slightly decreases this increase, but the location of accumulation shifts from the inner membrane to the outer membrane, implying a decreased cholesterol transfer from outer to inner membrane. Although the capacity of outer mitochondrial membranes was saturated after a 10-min treatment with CX, a 20-min treatment resulted in further retention of cholesterol in intact mitochondria that was not recovered in the isolated membranes. An additional pool of loosely bound cholesterol is proposed for CX mitochondria. These studies provide evidence that the CX-sensitive step of adrenal steroidogenesis attributed to loss of a labile ACTH regulatory protein (Pedersen, R.C. and Brownie, A.C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1882-1886) involves cholesterol transfer from the outer to the inner mitochondrial membrane. ACTH also enhances the PI and PE content of the outer membranes by a CX-sensitive mechanism that may contribute to intramitochondrial cholesterol transport. CX treatment does not affect cholesterol uptake by the inner membrane from phospholipid vesicles. The initial rate of endogenous metabolism in isolated inner membranes is insensitive to pretreatment (2 nmol/nmol P-450/min). The duration of this linear rate was increased 4-fold by AMG treatment while this increase was prevented by CX treatment. The kinetics indicate differences in inner membrane reactive cholesterol levels. Inner membranes also contained a fraction of unreactive cholesterol that is insensitive to pretreatment. Cholesterol-P-450scc complex formation for all pretreatments fits a single hyperbolic function of the reactive cholesterol content of the inner mitochondrial membrane (Kd = 0.025 mol cholesterol/mol phospholipid), and is activated over 5-fold upon mitochondrial disruption. All changes in inner membranes caused by CX can, therefore, be attributed solely to the restricted cholesterol access in vivo.

Assay of the high-alkaline proteinase from alkalophilic bacillus PB92 using a chromogenic tripeptide substrate.

The chromogenic tripeptide substrate, benzyloxycarbonylglycyl-L-prolyl-L-citrulline p-nitroanilide, is proposed for the assay of the high-alkaline proteinase, HAP-PB92, from an alkalophilic Bacillus. The assay method is sensitive, reproducible, and may be adapted for an automatic system.

Nutrition, education and fertility behaviour: rural Amritsar.

PIP: This study was undertaken to assess the influence of socioeconomic factors on fertility behavior in the rural Punjab, an area where a broad based rural development strategy has led to rapid socioeconomic change in the past 1 1/2 decades. 350 ever married women and their husbands from rural areas of Amritsar, Punjab, were interviewed in 1977. The number of births was interpreted as a linear function of 9 independent variables: husband's and wife's education, per capita calorie intake, per capita housing use, husband's caste, profession, per capita income, number of years married, and husband's and wife's ages at marriage. Average years of education of husbands was 4 years, while among wives it was 2 years. The average monthly per capita income of Rs. 146 and average per capita calorie intake of 2784 were significantly above the national averages. Average age at marriage was just over 22 for males and just over 19 for females. The average number of births/couple was 5. Husband's education greater than 10 years and wife's education greater than 8 years maximized the fit and were used in the equations. Because of the strong correlation between husband's education and per capita income, 2 alternative regressions were run, 1 with the education and 1 with the income variable. In the 1st regression, the coefficients for husband's education, wife's education, per capita calorie intake, number of married years, and husband's age at marriage were significant at the 1% level, the coefficient for wife's age at marriage was significant at the 5% level, and that for caste was significant at the 10% level. The F value was 58.75 and over 62% of the variation in the number of births was explained by the independent variables. The coefficients in the 2nd equation were also significant except that for per capita income. The coefficient for housing factors, which was not significant in the 1st equation, was significant at the 5% level. Per capita calorie intake was the most important single variable; it was estimated that an 11% increase in the per capita calorie intake would lead to about a 33% decline in births.^ieng