John P. Jones

Effect of vitamin K homologues on the conversion of preprothrombin to prothrombin in rat liver microsomes

Summary The vitamin K-dependent conversion of preoporthrombin to prothrombin has been studied in rat liver microsomes plus cytosol. Half maximal activities were obtained at pO2 of 10 mm Hg and 0.2 mM bicarbonate. A linear semi-logarithmic dose-response curve was obtained for K1, MK-3, and MK-4 over a range of 10−5 to 10−8 M. Other homologues showed a less extended range of response. Arrhenius plots show that the activation enthalpy for MK-2 was lowest with increasing enthalpies for MK-3, MK-4 and K1, the latter showing a conspicuous break in the curve corresponding to the “melting point” of the membrane.

The biosynthesis of cholesterol and other sterols by brain tissue: I. Subcellular biosynthesis in vitro

The biosynthesis of cholesterol by subcellular particles from rat brain was studied with several labeled cholesterol precursors as substrates. Rats from two age groups were used for preparation of the subcellular fractions: 15-day-old and adult. Microsomes and a soluble fraction were required for maximum biosynthesis of14C-nonsaponifiable material. The latter was synthesized in good yield by subcellular fractions from both age groups, but 90% or more was present as14C-squalene, when either U-14C-glucose, 2-14C-sodium acetate or 2-14C-mevalonic acid was the radioactive substrate. Neither3H-squalene oxide nor14C-lanosterol was converted to sterol when incubated with microsomal+soluble preparations, but some 4% of14C-desmosterol was converted to cholesterol by adult preparations. Thus a metabolic block, largely between squalene to desmosterol, exists in isolated microsomal+soluble preparations from both 15-day-old and adult rat brain.

RATE OF STEROL FORMATION BY RAT BRAIN GLIA AND NEURONS IN VITRO AND IN VIVO

The ability of 11‐day‐old rat glial and neuronal cells to biosynthesize sterol was studied as a function of time in vivo and in vitro. The in vitro experiments utilized [2‐14C]mevalonic acid as precursor. Glial‐enriched cell preparations demonstrated a greater ability to incorporate [2‐14C]mevalonic acid into isoprenoid material than did neuronal‐enriched preparations. Approximately 4 h were required for maximal uptake of labelled mevalonate by the glial preparations. Further metabolism of the isoprenoid material, involving squalene turnover and sterol demethylation, was still evident even after 15 h of incubation. In vivo, sterol biosynthesis was studied by intraperitoneal injection of sodium [2‐14C]acetate and [U‐14C]glucose, sacrifice of the animals at 2 or 24 h, subsequent isolation of glial‐ and neuronal‐cell enriched fractions and analysis of labelled isoprenoid material. Glial‐enriched fractions again contained the bulk of the labelled isoprenoid material.

Lipid biosynthesis in neuron-enriched and glial-enriched fractions of rat brain: Ganglioside biosynthesis

Abstract The incorporation of DL [3- 14 C]serine and N-acetyl [4- 14 C] neuraminic acid into neuron-enriched and glial-enriched lipid fractions was studied. The neuronal-enriched fraction incorporated 14 C-label into gangliosides to a greater extent than the glial-enriched fractions, substantiating previous indications that the neurons are the principal site of ganglioside biosynthesis in the brain.

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE.: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INTRACEREBRAL INJECTION OF [2-14C]-MEVALONIC ACID

Abstract— The distribution of 14C into several subcellular fractions of adult rat brain was studied as a function of time, following intracerebral injection of [2‐14C]mevalonic acid. As expected from previous studies, the microsomal fraction was indicated as the site of sterol biosynthesis. The myelin fraction showed a marked and early uptake of I4C‐labelled, digitonin‐precipitable material. This was assumed to be a non‐enzymic uptake of sterol intermediates. The mitochondrial fraction exhibited a rapid uptake of 14C‐labelled, nonsaponifiable material, but a very slow accumulation of 14C‐labelled, digitonin‐precipitable product. Examination of the nonsaponifiable 14C‐fractions by TLC showed a rapid appearance of labelled 4‐desmethyl sterols in the microsomal fraction. The myelin fraction selectively retained 4,4′‐dimethyl sterol but seemed to release this with time, possibly to be further metabolized by the microsomes. Examination of [14C]digitonin‐precipitable material by the dibromide method showed that although labelled 4‐desmethyl sterol appeared quite early, cholesterol itself was formed slowly in all fractions.

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [14C]labelled material into subcellular fractions of 30‐day‐old rat brain was studied as a function of time, following intracerebral injection of [2‐14C] mevalonic acid. As in the adult and 15‐day‐old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15‐day‐old animal, the myelin fraction from the 30‐day‐old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [14C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non‐myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non‐myelin to myelin fractions at about 37–44 days of age.

Lipid biosynthesis in neuron-enriched and glial-enriched fractions of rat brain: Sterol formation

Abstract Glial-enriched fractions were shown to synthesize sterol from [2- 14 C] mevalonic acid. Neuron-enriched fractions incubated with [2- 14 C] mevalonic acid, formed much less labeled neutral lipid than did glial-enriched fractions incubated under identical conditions. Sterol esters were produced only in the presence of neuron-enriched fractions. Labeled squalene and lanosterol were identified in the neutral fraction from glial-enriched incubations.

Isolation and characterization of isoprothrombin in the rat.

Abstract Isoprothrombin, a protein antigenically related to prothrombin, which accumulates in the hepatic endoplasmic reticulum of vitamin K-deficient or warfarin-treated rats, has been isolated by affinity chromatography employing rat prothrombin antibodies linked to Sepharose. Isoprothrombin has the same molecular weight as prothrombin but a different mobility on disc gel electrophoresis, is not barium adsorbable nor activatable to thrombin by factor Xa. Isoprothrombin is converted to thrombin by Echis carinatus venom through the same intermediates as prothrombin.

The biosynthesis of cholesterol and other sterols by brain tissue. Distribution in subcellular fractions as a function of time after intracerebral injection into developing brain of (2- 14 C)mevalonic.

Abstract— The distribution of [14C]‐labeIled material into subcellular fractions of 15‐day‐old rat brain was studied as a function of time after intracerebral injection of [2‐14C]mevalonic acid. As previously shown for adult brain, the data indicated the microsomal fraction to be the site of sterol biosynthesis. The synaptosomal fraction exhibited a marked early uptake of [14C]‐nonsaponifiable material. Total radioactivity in both myelin and myelin‐like fractions remained low in comparison to that in the other subcellular fractions at all time periods examined. At 2 h after injection, labelled digitonin‐precipitable material was demonstrable in all subcellular fractions. Examination of the [14C]‐labelled nonsaponifiable material by thin‐layer chromatography indicated the rapid appearance of labelled 4‐desmethyl sterol in all subcellular fractions, with the most rapid appearance in the myelin fraction, followed in decreasing order by microsomal, synaptosomal, and mitochondrial fractions. Examination of [14C] digitonin‐precipitable material from each fraction by the dibromide method demonstrated that although 4‐desmethyl sterol appeared quickly, the formation of cholesterol was slow in all fractions, an effect that had been reported earlier for adult brain.

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE: DISTRIBUTION IN SUBCELLULAR FRACTIONS AS A FUNCTION OF TIME AFTER INJECTION OF [2‐14C]MEVALONIC ACID, SODIUM [2‐14C]ACETATE AND [U‐14C]GLUCOSE INTO 15‐DAY‐OLD RATS

The distribution of [14C]‐labelled material into subcellular fractions of 15‐day‐old rat brain was studied at 2 and 24 h following intraperitoneal and intracerebral injection of [2‐14C]sodium acetate, [U‐14C]glucose and [2‐14C]mevalonic acid respectively. The total quantity of labelled isoprenoids in the brain was, except for glucose, greater when the precursor was administered intracerebrally. The intraperitoneal route was more advantageous in the case of [U‐14C]glucose. The subcellular distribution of both labelled total isoprenoid material and sterol was distinct for each labelled precursor. Intracerebrally injected [U‐14C]glucose at both time periods studied suggested no dominance of labelling in any fraction. After intraperitoneal injection of [U‐14C]glucose the microsomes were more prominently labelled. Both methods of administration of sodium [2‐14C]acetate resulted in heavy labelling of the myelin fraction after 24 h. The total labelled isoprenoids resided mainly in the microsomes 24 h after injection of [2‐14C]mevalonic acid. Labelled sterol was found to be localized more in the myelin and microsomal fractions for all three precursors than was the labelled total isoprenoids. Depending on the type of experiment to be conducted, each of these precursors can give different results, which must be interpreted accordingly.