The metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to 35 S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/10 6 cells) sustained activated cholesterol metabolism (0.4 pmol/min/10 6 cells, t1 ⁄2 ‫؍‬ 70 min) long after complete removal of p37 (t1 ⁄2 ‫؍‬ 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism.The conversion of cholesterol to pregnenolone, the first step in steroid synthesis, is catalyzed by cytochrome P450 side chain cleavage enzyme (P450scc), 1 which is localized on the matrix side of the inner mitochondrial membrane (1, 2). The transfer of cholesterol to P450scc, a limiting step in this conversion (3, 4), requires hormonal activation of cholesterol mobilization to the mitochondria. This mobilization is dependent on the uptake of lipoproteins, lysosomal-cytosolic transfer, and hydrolysis of cholesterol esters as well as on two subsequent transfer steps (5). The first step involves transfer of cholesterol to the mitochondria and is dependent on an intact cytoskeleton but not on protein synthesis (6). The second step involves transfer within the mitochondria from outer to inner membrane. This trophic hormone-dependent transport of cholesterol from mitochondrial outer membrane to inner membrane (4, 7, 8) is blocked in vivo within 10 min by protein synthesis inhibitors, such as cycloheximide (CHX) (9, 10). A series of cAMP-stimulated and CHX-sensitive phosphoproteins (30 -37 kDa), which localize to the mitochondria, have been identified in cultured adrenal and testis cells (11)(12)(13)(14)(15)(16)(17). The gene that encodes these proteins has been cloned, and the active form has been named the steroidogenic acute regulatory protein (StAR) (18). The exp...

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In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).

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