5$\alpha$-Cholest-8(14)-en-3$\beta$-ol-15-one is a potent inhibitor of cholesterol biosynthesis in cultured cells. Cells derived from the CHO K-1 cell line have been isolated based on their ability to grow in high concentrations of 5$\alpha$-cholest-8(14)-en-3$\beta$-ol-15-one (up to 40 $\mu$M). These cells, designated as the 15-ketosterol-adapted or K(15) cells, accumulate cytoplasmic lipid-rich regions that appear as inclusions when observed by light microscopy. Results of studies presented herein show that K(15) cells incubated at 40 $\mu$M 5$\alpha$-cholest-8(14)-en-3$\beta$-ol-15-one incorporated almost seven times as much of the 15-ketosterol as K(15) cells incubated at 15 $\mu$M 15-kestosterol. Upon incubation of K(15) cells in lipid-rich medium containing 40 $\mu$M (2,4-$\sp3$H) 5$\alpha$-cholest-8(14)-en-3$\beta$-ol-15-one, $\sim$96% of the radioactivity recovered in an isolated floating lipid layer corresponded to 15-ketosteryl esters. Upon the addition of sodium (1-$\sp{14}$C) oleate (82 $\mu$M) to the incubation medium, the radioactivity recovered in the floating lipid layer corresponded to both 15-ketosteryl esters and triacylglycerols. Following removal of the 15-ketosterol from the growth medium, the K(15) cells excreted 66-88% of the incorporated 15-ketosterol into the medium.

High resolution $\sp{13}$C NMR was used to study the formation of metabolites from (1-$\sp{13}$C) -labelled glucose by wild-type and HOG (high osmolarity glycerol response) mutants of the yeast Saccharomyces cerevisiae after transfer to media containing 400 mM NaCl. Time course spectroscopy showed that the major end-products were (2- $\sp{13}$C) -ethanol, (1-$\sp{13}$C) -glycerol and in much lower concentration, (2-$\sp{13}$C) -acetate. High osmolarity increased ethanol, glycerol and acetate production. Additional labeling incorporated in trehalose, glycogen or glutamate was similar in all strains. In contrast to the wild-type, the hog1 mutant decreased glycerol and acetate production in normal and hyperosmotic media, whereas the hog2 mutant only reduced glycerol synthesis under high osmolarity. The decreases in osmolarity-induced glycerol synthesis were quantitatively additive in the hog1 hog2 mutant. Effects of the hog1 and hog2 mutations on yeast fermentation suggest that the HOG1 and HOG2 genes regulate or influence different aspects in fermentation metabolism.

A simple, rapid method for the purification to near-homogeneity of rat skeletal muscle adenylosuccinate synthetase was developed. Using enzyme prepared by this method, the molecular weight of the native protein was determined to be approximately 100,000 daltons and several kinetic parameters were evaluated. The kinetic properties of the enzyme indicate that it might be regulated in muscle primarily by the availability of its substrate, inosine-5'-monophosphate. Under some conditions, inhibition by guanosine-5'-diphosphate and by fructose 1,6-diphosphate may also be factors. These properties are consistent with the proposed role of the enzyme in the purine nucleotide cycle in muscle. The activities of the two adenylosuccinate synthetase isozymes in rat liver were measured under varying dietary conditions. The two isoenzymes are separable by ion-exchange chromatography, thus affording a rapid means of analysis. It was found that the expression of the acidic isozyme appears to be coordinated with purine biosynthesis, while the activity of the basic isozyme increases in situations where the animal is metabolizing large amounts of protein. These results indicate that the acidic isozyme is regulated for biosynthesis, while the basic isozyme may be associated with the purine nucleotide cycle. Total activities of adenylosuccinate synthetase and lyase did not change significantly. Since the adenylosuccinate synthetase isozyme content of a tissue might serve as a useful indicator of the direction of metabolism in that tissue, the relative amounts of the two were measured in several different organs. It was found that, under normal conditions, all organs tested contained some of the basic isozyme; it was the only form detectable in skeletal muscle and heart. Liver, kidney, and brain contained both types. In liver the two activities were present in roughly equal amounts, while the acidic form predominates in kidney and brain. The effects of long-term changes in dietary regimen on hepatic adenine and guanine nucleotide pools and de novo purine synthesis and on serum allantoin levels in growing rats were determined. It was found that dietary guanine had relatively little effect on hepatic guanine metabolism, but that adenine is incorporated directly into liver adenine nucleotide pools. High protein and carbohydrate diets also increased the soluble adenine level. The rate of de novo synthesis appears to be unaffected by changes in nucleotide concentration, but is in some way decreased by long-term feeding of large amounts of protein or carbohydrate, indicating that steady-state adaptations to changes in dietary regimen may not follow the same patterns as have been proposed for short-term conditions.

Cibacron Blue 3G-A substituted on an agarose or dextran matrix has become widely used as an absorbant for enzyme purification. Its mode of interaction with the proteins which bind to it is not entirely clear. One popular theory for proteins' attraction for the dye is that it is a specific structural probe for the supersecondary protein structure called the dinucleotide fold (Stellwagen E. (1977) Acct. Chem. Res. 10, 92-98). The purpose of this investigation is to more clearly define this dye's mode of affinity for proteins. It has been determined through both competitive inhibition kinetic studies and the use of dyed agarose matrixes that the major portion of Cibacron Blue 3G-A that is involved in its binding to the selected dehydrogenases and kinases studied is 1-amino-4(4'-aminophenylamino)anthraquinone-2,3'-disulfonic acid. Effective interaction of a structural isomer and smaller moieties of Cibacron Blue 3G-A with these selected kinases and dehydrogenases indicate a largely nonspecific interaction with respect to the exact structure and conformation of the dye. There is much evidence available that a combination of hydrophobic and electrostatic interactions are taking place at a hydrophobic patch or pocket on the enzymes. This interaction is not sufficiently specific to be used as a probe for a defined protein supersecondary structure. These studies have shown the potential of dyes for determining an enzyme's kinetic mechanism through competitive inhibition. The unusual inhibition patterns exhibited by Cibacron Blue 3G-A with phosphoglycerate kinase allowed a clear demonstration of the random mechanism of that enzyme. Two new columns were developed for use in the purification of mainly nucleotide requiring enzymes using dyes covalently bound to an agarose matrix. These columns represent the feasibility of developing a large number of new chromatographic materials that are inexpensive, easily prepared, and offer a wide variability.

In contrast to the analogous reaction with xanthine the anaerobic rapid reduction of xanthine oxidase by excess lumazine (2,4-dihydroxypteridine) displays markedly biphasic kinetics at all wavelengths. The fast phase proceeds at a rate (30 s('-1)) approximately a factor of 100 faster than that of the slow phase and about 50 times more rapid than catalysis. During the rapid phase the decrease in absorbance at 450 nm is approximately equal to the corresponding increase at 650 nm ((DELTA)(epsilon) = 6400 M('-1)cm('-1)). The presence of the rapid increase in absorbance at 650 nm during the analogous reduction of deflavo-xanthine oxidase demonstrates that this spectral component is not a function of the flavin. The slow phase shows a decrease in absorbance at all wavelengths proceeding at a rate of 0.3 s('-1). At the conclusion of this anaerobic reduction with lumazine a net increase in absorbance above 600 nm is observed. The long wavelength absorbance results from the interaction of the end-products of the anaerobic reduction, i.e. Mo(IV) and violapterin (2,4,7-trihydroxypteridine), which gives rise to a charge transfer complex. Circular dichroism studies demonstrate that the charge transfer complex is optimically active. A linear Scatchard plot obtained for the titration of dithionite-reduced enzyme with violapterin yields an apparent dissociation constant of 7.9 (mu)M for the reduced enzyme-product complex. However, the complementary rapid kinetic study of this enzyme-product interaction displays a biphasic time course-indicating that this binding process proceeds by a complex mechanism. Mixing dithionite-reduced xanthine oxidase with lumazine results in the formation of a charge transfer complex similar to that found with violapterin. No other pteridine tested displays this long-wavelength absorption when mixed with the dithionite-reduced enzyme although a rapid increase in absorbance at 650 nm is observed during the reduction of xanthine oxidase by pterin or xanthopterin. Inactivating the enzyme with either cyanide or allopurinol prevents the formation of the charge transfer absorbance. The anaerobic reduction of xanthine oxidase by lumazine can be observed by both absorbance and fluorescence spectroscopy. Following the reduction of the enzyme at 450 nm, the formation and subsequent breakdown of the enzyme-product complex at 650 nm, and monitoring the conversion of lumazine to violapterin via fluorescence the kinetics of the reaction can be defined. There appears to be a good correlation between these four optical probes. At pH 8.3 the formation of the enzyme-product complex occurs at a rate of 51 s('-1) while the subsequent breakdown proceeds at 0.4 s('-1). The rate of the former step depends on the absolute concentration of the substrate and an apparent dissociation constant for the enzyme-substrate complex of 34 (mu)M is obtained. The rate of the latter process is independent of the concentration of the substrate although this rate does appear to be affected by the stoichiometry of the reaction. These results are consistent with the model of Olson, J.S., Ballou, D., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382 formulated for the analogous study with xanthine. Finally, there exists a reasonably good correlation between the kinetic parameters obtained from the rapid kinetic studies and those determined for the complementary steady-state results. The effect of pH on the reaction of xanthine oxidase with lumazine mimics that found for the analogous reaction with xanthine. These studies lead to a more general description of the reaction mechanism of xanthine oxidase.

The nonspecific DNA binding capacity of the lac repressor protein has been assessed by two different methods. Boundary sedimentation of repressor and calf thymus DNA fragmented by shearing yielded dissociation constants in good agreement with values previously reported in the literature. The b

The sterol composition of bovine retinas and rod outer segment (ROS) membranes was examined. Cholesterol accounted for >98% of the total sterol and (TURN)2% of the dry weight of both the retina and ROS membranes. Minor amounts of components having the chromatographic properties of 5(alpha)-cholestan-3(beta)-ol and 5(alpha)-cholest-7-en-3(beta)-ol were detected in whole retinas, and a "cholestanol-like" component was also detected, in minor amounts, in ROS membranes. Calculations indicated that the molar ratio of cholesterol:rhodopsin in ROS membranes was (TURN)5. Using literature values for the phospholipid content of bovine ROS membranes, the calculated cholesterol:phospholipid molar ratio was (TURN)0.06 and cholesterol only represented (TURN)5-7 mole % of the total ROS membrane lipid. The metabolism of ('3)H-labelled mevalonic acid was studied in vitro with "intact" retinas and with the 10,000 x g supernatant (S(,10)) fraction. The major nonsaponifiable products obtained from incubations of "intact" retinas had the chromatographic properties of squalene and lanosterol; only minor amounts of label were incorporated into material having the chromatographic properties of C(,27) monohydroxy sterols. The retinas also converted mevalonic acid to isoprenoid acids; no label was incorporated into retina n-fatty acids. In the incubations with the S(,10) fraction, <1% of the label incorporated into nonsaponifiable lipids was precipitable with digitonin. The major nonsaponifiable lipids had the chromatographic properties of squalene and various open-chain isoprenoid alcohols, but did not correspond to the alcohols of the allyl pyrophosphates which are known intermediates in the biosynthesis of squalene. Material having the chromatographic properites of C(,30) and C(,27) monohydroxy sterols was detected in small amounts in these incubations, but cholesterol represented only a minor fraction of this material. Isoprenoid acids (primarily C(,20) isomers, with lesser amounts of C(,15) isomers) were formed as the major products of these incubations. In addition to material having the properties of the all-trans isomers of farnesoic acid and geranylgeranoic acid, the biosynthesis of compounds having the chromatographic properites of the cis,cis- and cis,trans- (or trans,cis-) isomers of farnesoic acid was noted. Also, relatively large amounts of a C(,20) isomer of geranylgeranoic acid (apparently having at least one cis double bond) were recovered from such incubations. These isoprenoid acids were not detected as endogenous components of the retina. In an in vivo experiment, intraocularly-injected ('14)C-labelled mevalonic acid was taken up and metabolized to nonsaponifiable lipids by several ocular tissues (predominantly by the retina) over a period of up to 2 hours. In each tissue, the major labelled nonsaponifiable lipid was squalene, with lesser amounts of material having the chromatographic properties of lanosterol. Only a few per cent of the retina nonsaponifiable products behaved chromatographically like C(,27) monohydroxy sterols. It was inferred from these results that the bovine retina has a very limited capacity for de novo sterol biosynthesis and must rely on alternate sources of cholesterol for the biogenesis of ROS membranes.

Cytochrome oxidase, the terminal oxidase of the electron transport chain, is classically known to be a membrane bound protein of a molecular weight close to 200,000 Daltons which contains two copper atoms (Cu(,D) and Cu(,U)) and two heme groups, cytochromes a and a(,3). The enzyme is found in aerobic organisms where its function is to catalyze the reduction of oxygen to water and to promote oxidative phosphorylation to satisfy the energy requirements of the cell. The purpose of this thesis is to define the oxidative-reductive (redox) intermediates of the enzyme. Physico-chemical methods are used to obtain information on the structural and valence characteristics, the electronic states, and the redox properties of the four metals involved in catalysis. Methods such as magnetic susceptibility, electronic absorption spectroscopy, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MCD) have been used to monitor several types of redox intermediates. Redox titrations have been performed under different pH conditions, with various ligands, with several mediators, under variable salt concentrations, and in the presence of mitochondrial metabolites such as ATP. The low temperature (77(DEGREES)K) MCD spectra of "mixed-valence" cytochrome oxidase have been recorded. By using multiple linear regression of the spectral data and computer simulations of the redox titrations monitored by optical EPR, and MCD spectroscopies the absolute and relative redox potentials of the enzyme under various conditions have been obtained. With these methods the redox intermediates of cytochrome oxidase can be defined. The results indicate that for the reductive titration in the presence of cytochrome c at pH 7.4 (15 C) the potentials are: cytochrome a = cytochrome a(,3) = Cu(,U) = 314 mV, Cu(,D) = 272 mV. At pH 8.5 (15 C), the potential of cytochrome a(,3) is lowered by 13 mV and the potential Cu(,U) is lowered by 38 mV with respect to cytochrome a. In the presence of inhibitors which stabilize the oxidized form of cytochrome a(,3) (azide and formate), the redox potentials of cytochrome a(,3) and Cu(,U) are lowered by 77 mV with respect to cytochrome a. In the presence of inhibitors which stabilize the reduced form of cytochrome a(,3) (carbon monoxide), the redox potential of cytochrome a(,3) is increased by 100 mV. No evidence of "heme-heme interaction" is observed at equilibrium. The relative potentials measured at 15 C and at cryogenic temperatures indicate that electron rearrangements are occurring among the redox centers of the partially reduced enzyme samples during freezing of the EPR samples. A model for the bridging between cytochrome a(,3) and the Cu(,U) center is presented which explains the three types of high-spin signals that are observed. This thesis clarifies the assignments of the electronic and magnetic characteristics which each redox center exhibits, defines the absolute and relative potentials associated with each metal center, and discriminates between the existing models of the mechanism of cytochrome oxidase in order to establish the oxidative-reductive mechanism of the enzyme.

5(alpha)-Cholest-8(14)-en-3(beta)-ol-15-one at 10('-4) M has been shown to inhibit the conversion of acetate, but not mevalonate, to nonsaponifiable lipids and digitoninprecipitable sterols by 10,000 x g supernatant preparations from rat liver homogenates. In addition, a variety of structurally related compounds were tested in this system. The direct addition of 5(alpha)-cholest-8(14)-en-3(beta)-ol-15-one to various cell-free preparations had no effect on the individual enzymes catalyzing the conversion of acetate to mevalonate. The administration of dietary 5(alpha)-cholest-8(14)-en-3(beta)-ol-15-one (0.1%) to male rats for eight days caused a significant decrease in serum cholesterol levels and an increase in the hepatic activities of acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase. 14(alpha)-Ethyl-5(alpha)-cholest-7-en-3(beta),15-diol when administered by daily subcutaneous injections in triolein to male rats maintained on a Cholesterol-Free Test Diet has been shown to cause a 23 percent reduction in serum cholesterol levels after 13 days. A variety of oxygenated sterols containing a 15(alpha)-hydroxyl group were found to inhibit the conversion of acetate and mevalonate into digitonin-precipitable sterols by 10,000 x g supernatant preparations from rat liver homogenates. The structural parameters important in this inhibitory effect have been investigated. Cycloheximide at 10('-3) M has been found to inhibit the synthesis of digitonin-precipitable sterols from acetate, but not mevalonate, in 10,000 x g supernatant fractions of rat liver homogenates. This effect was not due to an inhibition of protein synthesis. The deprivation of food for 48 hours has been shown to cause a reduction in the activities of acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase in rat liver.

Part 1. 5(alpha)-Cholest-8(14)-en-3(beta)-ol-15-one, prepared by chemical synthesis, was administered to male rats at a concentration of 0.1% in a low ((TURNEQ)0.005%) cholesterol diet. These rats showed a sustained decrease in food consumption and a concomitant decrease in body weight. The me