A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

Decrease of estrogen receptors induced by 17 beta-estradiol and progesterone in cultured rabbit endometrial cells

A whole cell technique to measure estradiol receptors in cultured rabbit endometrial cells is described. The estradiol receptors measured appear to be composed of at least two components: one component has high affinity but low capacity, while the other component has low affinity but high capacity. Using the assay, the effects of estradiol and progesterone pretreatment were examined on the estradiol receptor levels. It was found that both of the hormones decreased the number of estrogen receptors in the cultured cells. The finding that estradiol decreased its own receptors was unexpected and its possible relevance is discussed.     

A spectrofluorimetric investigation of calf thymus DNA modified by BP diolepoxide and 1-pyrenyloxirane

7β,8α-Dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP diolepoxide, $\text{1}$) and 1-pyrenyloxirane ($\text{2}$) bind chemically to calf thymus DNA. The fluorescence efficiency of pyrenyl groups in mutagen modified DNA varies appreciably with its conformation and decreases in the order: pyrenees, modified denatured DNA and modified native DNA. A particularly interesting observation is that the fluorescence efficiency of mutagen modified DNA intensifies substantially upon denaturation. Our results suggest that the pyrenyl groups in mutagen modified DNA are intercalated between the base pairs of DNA. Since both $\text{1}$ and $\text{2}$ are powerful frame-shifting mutagens for S. typhimurium TA-98, the intercalative covalent binding of these compounds to DNA may provide a molecular basis for their mutagenic activity.     

Studies on the red oxidase (cytochrome o) of Azotobacter vinelandii

In an attempt to isolate and to study the electron transport system of $\text{Azotobacter vinelandii}$, we have isolated and purified a membrane-bound cytochrome $\text{o}$. The cytochrome $\text{o}$, purified as a detergent (Triton X-100) and hemoprotein complex, contained 1.6 nmoles heme per mg of protein. Cold-temperature spectrum showed that no other cytochrome was associated with the purified preparation, and electrophoresis revealed that only one type of hemoprotein was obtained. The purified cytochrome $\text{o}$ reacted with both carbon monoxide and cyanide readily. Only in the reduced form did it combine with carbon monoxide, whereas the oxidized form reacted with cyanide. An “oxygenated” form of the cytochrome $\text{o}$ was demonstrated to be spectrally distinguishable from both the oxidized and the reduced forms.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes.

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor

Summary 80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR ⁺ strain but not by corresponding preparations from an argR ^- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on -galactosidase synthesis.     

In vivo inhibition of cholesterol uptake in rabbit aortas by 7-ketocholesterol.

The iv injection of 7-ketocholesterol into rabbits, made soluble by combining with bile salts, inhibited cholesterol uptake by the aorta. However, the inhibition was not as marked or as uniform as previously demonstrated in in vitro experiments. This difference may have been the result of lower plasma concentrations of 7-ketocholesterol in the injected animals. Gastric feeding of 7-ketocholesterol failed to inhibit aortic cholesterol uptake, probably because of inadequate plasma concentrations of the inhibitory steroid. The results suggest that the mechanism of 7-ketocholesterol on aortic cholesterol uptake is through competitive inhibition.     

Utilization of cellulosic materials through enzymatic hydrolysis. II. Preliminary assessment of an integrated processing scheme

An integrated processing scheme is described for the conversion of a cellulosic waste (newsprint) to sugars by enzymatic hydrolysis and then to ethanol and yeast by fermentation. The unconverted solids are burned to produce process energy requirements and surplus electrical power. Preliminary designs and cost studies are developed to provide a rough perspective on the potential economic feasibility of this method of cellulose utilization.     

Generation of ATP by chloroplasts through solvent perturbation.

A two-stage method was discovered for generating ATP by chloroplasts in the dark at constant pH through solvent perturbation. With cold acetone as the perturbing solvent, the yield of ATP was found to increase with the volume percent of acetone in the first stage medium. The results are difficult to explain in term of the proton gradient model, but is consistent with the conventional model of prior water formation and subsequent ATP generation.     

Neutral lipids of Aedes aegypti and Aedes albopictus cells cultured in vitro.

Aedes aegypti feeding on chickens infected with Plasmodium gallinaceum take less blood and lay fewer eggs than those feeding on uninfected hosts. Both activities show an inverse correlation with the degree of parasitemia. Mosquitoes feeding on infected chickens ingest blood in amounts directly proportional to the length of time spent on the hosts, whereas there is no relationship between host contact and blood meal size for mosquitoes feeding on uninfected hosts. Feeding and probing choice experiments demonstrate that infected chickens are less attractive to Aedes aegypti than uninfected chickens.