Application of commercial enzymes to measure the activity of the arginine pathway-urea cycle in intact cells

The activity of the complete arginine pathway-urea cycle was assessed in intact plant cells by employing the commercial enzymes arginase (EC 3.5.3.1) and urease (EC 3.5.1.5) to determine the amount of NaH14CO3 incorporated into [guanido-14C]arginine and/or into [14C]urea during a 3-h labeling period. Recovery of [guanido-14C]arginine was linear from 5 to 1000 nmol/g tissue and averaged 80 +/- 5% (mean +/- SE, N = 3). The procedure is reliable, inexpensive, well suited to the simultaneous analysis of numerous samples, and significantly more sensitive than existing methods. The method is ideally suited for assessing the activity of the complete arginine biosynthetic pathway in intact cells. In addition, the method has the distinct advantage of providing simultaneous measurement of the amount of NaH14CO3 accumulating in arginine relative to the amount accumulating as urea. Evidence is presented demonstrating that both the activity of the arginine pathway and the relative amounts of [guanido-14C]arginine and [14C]urea synthesized from NaH14CO3 were influenced by changes in the level of ornithine, NH+4, or phosphorus available to plant tissues.     

Stimulation by D-glucose of the direct conversion of arginine to citrulline in enterocytes isolated from pig jejunum

In enterocytes isolated from pig jejunum, L-arginine is metabolized to L-citrulline either directly or indirectly through the sequence of reactions catalysed by arginase and ornithine transcarbamylase. In the presence of 5 mM D-glucose, the direct conversion of 1mM L-[guanido-14C] arginine to L-citrulline was increased more than 4 times. Isolated enterocytes exhibit a high glycolytic capacity. Furthermore, the decarboxylation of 5mM D-[1-14C] glucose was 3.6 fold higher than the decarboxylation of 5 mM D-[6-14C] glucose which suggests the presence of a pentose phosphate pathway in enterocytes. Since the production of labelled L-citrulline from L-[guanido-14C] arginine in pig enterocyte homogenates was markedly increased in the presence of NADPH, it is proposed that the direct conversion of L-arginine to L-citrulline could be stimulated by the production of NADPH from D-glucose in the pentose phosphate pathway.     

Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed.     

Turnover of Glial Filaments in Mouse Spinal Cord

Twenty-day-old mice received a single tail vein injection of [guanido-14C]arginine. The cytoskeleton was extracted from the spinal cords at varying lengths of time thereafter. Glial fibrillary acidic protein (GFAP) formed a distinct, broad band that was widely separated from other protein bands in one-dimensional polyacrylamide gels. The purity of the GFAP band was verified by Western blot analysis of one- and two-dimensional electrophoretic patterns. In addition, enzyme-linked immunosorbent assay and quantitative Western blot analysis indicated that 95% of the total spinal cord GFAP was extracted in the cytoskeletal preparation. The specific activity of GFAP was obtained by eluting the protein from the cytoskeletal GFAP band in preparative one-dimensional gels. Specific activity reached a peak 2 h after injection with [14C]arginine. Forty percent of the incorporated radioactivity was still present in cytoskeletal GFAP at 9 weeks, indicating that a significant proportion of glial filaments turns over relatively slowly in vivo.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

Conversion of [U-14C]threonine into 14C-labelled amino acids in the brain of thiamin-deficient rats.

In confirmation of the findings of Gaitonde et al. (1974), a decrease in the brain concentration of threonine and serine, and an increase in glycine, were observed in rats maintained on a thiamin-deficient diet. Similar changes were found in the blood, and the concentration of several other amino acids in the blood decreased significantly. There was a correlation between the concentrations of threonine, serine, aspartate and asparagine in the brain and blood. In experiments in which [U-14C]threonine was injected into rats most of the radioactivity in the brain and blood of control rats was, as expected, in threonine in the acid soluble metabolites. In contrast, a considerable proportion of radioactivity was also found in other amino acids, namely glutamate, glutamine, aspartate, gamma-aminobutyrate and alanine, in the brain of thiamin-deficient rats. [U-14C]Threonine was also converted into 14C-labelled lactate and glucose, but the extent of this conversion was severalfold higher in thiamin-deficient than in control rats. This finding gave evidence of the stimulation in thiamin-deficient rats of the catabolism of [U-14C]threonine to [14C]lactate by the aminoacetone pathway catalysed by threonine dehydrogenase, and into succinate via propionate by the alpha-oxobutyrate pathway catalysed by threonine dehydratase (deaminase). The measurement of specific radioactivities of glutamate, aspartate and glutamine after injection of [U-14C]threonine, indicated a stimulation of the activities of threonine dehydrogenase and threonine dehydratase (deaminase) in the brain of thiamin-deficient rats. The specific radioactivities of glutamate, asparatate and glutamine int he brain were consistent with an alteration in the metabolism of threonine, mainly in the 'large' compartment of the brain of thiamin-deficient rats. The measurement of relative specific radioactivity of proteins after injection of [U-14C]threonine indicated a marked decrease in the synthesis of proteins, mainly in the liver of thiamin-deficient rats.     

Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet

We have shown that first-pass intestinal metabolism is necessary for approximately 50% of whole body arginine synthesis from its major precursor proline in neonatal piglets. Furthermore, the intestine is not the site of increased arginine synthesis observed during dietary arginine deficiency. Primed constant intravenous (iv) and intraportal (ip) infusions of L-[U-14C]proline, and iv infusion of either L-[guanido-14C]arginine or L-[4,5-3H]arginine were used to measure first-pass hepatic arginine synthesis in piglets enterally fed either deficient (0.20 g.kg(-1).day(-1)) or generous (1.80 g.kg(-1).day(-1)) quantities of arginine for 5 days. Conversion of arginine to other urea cycle intermediates and arginine recycling were also calculated for both dietary treatments. Arginine synthesis (g.kg(-1).day(-1)) from proline was greater in piglets (P < 0.05) fed the deficient arginine diet in both the presence (generous: 0.07; deficient: 0.17; pooled SE = 0.01) and absence (generous: 0.06; deficient: 0.20; pooled SE = 0.01) of first-pass hepatic metabolism. There was no net arginine synthesis from proline during first-pass hepatic metabolism regardless of arginine intake. Arginine conversion to urea, citrulline, and ornithine was significantly greater (P < 0.05) in piglets fed the generous arginine diet. Calculated arginine fluxes were significantly lower (P = 0.01) for [4,5-3H]arginine than for [guanido-14C]arginine, and the discrepancy between the values was greater in piglets fed the deficient arginine diet (35% vs. 20%). Collectively, these findings show that first-pass hepatic metabolism is not a site of net arginine synthesis and that piglets conserve dietary arginine in times of deficiency by decreasing hydrolysis and increasing recycling.     

Incorporation of label from d-β-hydroxy-[14C]butyrate and [3-14C]acetoacetate into amino acids in rat brain in vivo

The metabolism of ketone bodies by rat brain was studied in vivo. Rats starved for 48h were given either d-beta-hydroxy[3-(14)C]butyrate or [3-(14)C]acetoacetate by intravenous injection and killed after 3 or 10min. Total radioactivity in the acid-soluble material of the brain and the specific radioactivities of the brain amino acids glutamate, glutamine, aspartate and gamma-aminobutyrate were determined. A group of fed animals were also given d-beta-hydroxy[3-(14)C]butyrate. In the brains of all animals (14)C was present in the acid-soluble material and the specific radioactivity of glutamate was greater than that of glutamine.     

Effect of dietary level of protein on the metabolism of mouse liver nuclear proteins.

The effect of protein depletion and refeeding on the metabolism of mouse liver nuclear proteins was studied. Five days protein depletion caused a 35% decrease in total nuclear protein. A fast recovery of the lost proteins, except histones, was induced when depleted mice were refed with a normal diet. Depletion caused a decrease in total nuclear protein synthesis, whereas refeeding quickly restored its normal value. The rates of total nuclear protein breakdown were estimated either as the difference between synthesis and protein gain or from the decay of radioactivity in protein labeled by the administration of both sodium [14C]bicarbonate and [35S]methionine. By these procedures, it was found that refeeding caused a slowdown in total nuclear protein breakdown. Hence, the recovery of the protein content observed during refeeding is due to both a restoration of synthesis and a decrease of breakdown. The [14C]bicarbonate procedure did not permit to obtain a high efficiency of label and, therefore, it was unsatisfactory for the measurement of the breakdown of fractionated nuclear proteins. A labeling procedure using [35S]methionine was designed for adequate measures of the decay of radioactivity in these proteins. This allows us to find that a slow down in breakdown affects similarly during refeeding to histones, to non histones, and to a fraction which contains ribonucleoproteins and soluble proteins.     

The effect of cycloheximide on the glycosylation of lactating-rabbit mammary glycoproteins.

1. Cycloheximide inhibited immediately the incorporation of L-[4,5-3H]leucine and D-]2-3H]mannose into mammary proteins, suggesting that the mannosylation of mammary glycoproteins requires the continued supply of newly synthesized polypeptides. 2. The incorporation of radioactivity from N-acetyl-D-[1-14C] glucosamine into protein was not inhibited until approx. 30 min after cycloheximide addition. Much (greater than 90%) of this radioactivity was present as N-acetylgalactosamine. 3. N-Glycosylation appears to be inhibited immediately by cycloheximide due to a lack of newly synthesized acceptor polypeptides, whereas O-glycosylation continues for 30 min, the time taken for acceptor peptides to move from their site of synthesis to the Golgi region and for completion of glycosylation. 4. There was a transient increase in the incorporation of mannose into lipid-linked oligosaccharide in the presence of cycloheximide, followed by a decrease in the radioactivity in this fraction. 5. The major lipid-linked oligosaccharide extracted from explants incubated for 2h in the presence of cycloheximide (6-7 monosaccharide units) was smaller than that extracted from control explants (10-12 monosaccharide units).     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

Turnover of plasma membrane polypeptides in nonproliferating cultures of Chinese hamster ovary cells and human skin fibroblasts.

When cultures of Chinese hamster ovary cells were maintained in stationary phase on medium deficient in l-isoleucine (A) or low in serum (B), active protein turnover occurs. These cells can be acetylated with trace levels of radioactive acetic anhydride in order to incorporate label into all of the major species of polypeptides of the plasma membrane. Four days following acetylation with [³H]acetic anhydride and removal from medium A containing l-[¹⁴C]leucine, the specific ³H and ¹⁴C radioactivities of the plasma membrane proteins had fallen 15- and 7-fold respectively. The lower value obtained with the radioactive leucine is probably due to reutilization of this amino acid. The ³H and ¹⁴C radioactivity profiles for the polypeptides separated by discontinuous gel electrophoresis, however, showed little qualitative change over the course of the experiment, suggesting that differential rates of protein turnover were not occurring. These results were confirmed in experiments with cells using both the above culture conditions in which two acetylations were carried out, one with ³H at time zero and the other with contrasting ³C label up to 96 h later. Two methods for plasma membrane isolation and a number of electrophoretic conditions were employed. Again, however, the radioactivity profiles along the gels coincided almost exactly, even though the ³H specific radioactivity had fallen several fold. Similar results have been obtained with confluent human skin fibroblasts. We suggest that the major proteins in the plasma membranes of cultured mammalian cells do not show markedly heterogeneous rates of turnover. In particular, larger species of polypeptides do not appear to have shorter half-lives than smaller ones.     

Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations

A purified fraction of unstacked thylakoid membranes (TMF1u) has been obtained from homogenates of Chlamydomonas reinhardtii (wild type 137+) by using repeated centrifugates in sucrose density gradients and low salt treatment. The contaminants of the fraction are reduced to a few mitochondria (approximately 3% of the total mitochondrial population), a few osmiophilic granules, and fragments of chloroplast envelopes. By SDS-polyacrylamide gel electrophoresis the polypeptide components of TMF1u were resolved into at least 30 bands. To determine the relative rates of assembly of newly synthesized polypeptides into thylakoid membranes, synchronized algal cells were doubly labeled in vivo with L-[14C] and L-[3H]arginine--used for long- and short-term labeling, respectively. TMF1u's were isolated from the labeled cells at selected time points during the cycle and the distribution of radioactivity was assayed in the gel electrophoretograms of their solubilized polypeptides. Incorporation of newly synthesized polypeptides into the bands of the gels was found to occur continuously but differentially throughout the cycle. Maximal rates of incorporation for the majority of the polypeptides were detected shortly after cell division (6D-7D; equivalent to early G1 phase). The rates of radioactive labeling decreased gradually to a low level at the end of the dark period and then rose slightly at the beginning of the next light period. The findings suggest that, in addition to the light/dark control postulated in the past, assembly of newly synthesized proteins into thylakoid membranes is activated by signals at work in the early G1 phase.     

Cytokeratin 13 contains O-glycosidically linked N-acetylglucosamine residues

The glycosylation of human cytokeratins was investigated in cultured human keratinocytes and A431 cells by metabolic labeling with [3H]glucosamine. In the presence of tunicamycin, keratinocytes incorporated [3H]glucosamine into a vitamin A-regulated acidic 53-kDa component of the cytoskeleton which was identified as cytokeratin 13 by one- and two-dimensional immunoblotting with specific monoclonal antibodies. This cytoskeletal component was also labeled with [3H]glucosamine in A431 cells but not in KB cells, which do not express cytokeratin 13. Its labeling was resistant to tunicamycin, suggesting that [3H]glucosamine had not been incorporated into N-linked oligosaccharides. Acid hydrolysis followed by paper and ion-exchange chromatography showed that the radioactivity in electrophoretically purified cytokeratin 13 was still present as glucosamine. Radioactivity was completely removed by treatment with beta-N-acetylglucosaminidase, suggesting that it was present in terminal N-acetylglucosamine residues. The labeled carbohydrate was released by alkaline borohydride treatment and was bound by a phenylboronic acid column, indicating an O-glycosidic linkage. On Bio-Gel P-2 columns, the beta-eliminated carbohydrate co-eluted with authentic N-acetylglucosaminitol. The results indicate that cytokeratin 13 contains single residues of N-acetylglucosamine O-glycosidically linked to the polypeptide chain.     

The distribution of glucose and [14C]glucose between erythrocytes and plasma in the rat

1. Of the glucose in rat blood 79.8+/-3.3% (s.d.) was in the plasma. The variance was mostly due to differences between rats. 2. The concentration of glucose in erythrocyte water was 51+/-8% (s.d.) of that in plasma water. 3. The ratio (specific radioactivity in plasma)/(specific radioactivity in whole blood), i.e. the P/B ratio, was estimated for glucose at intervals after intravenous injection of [U-(14)C]glucose and [U-(14)C]fructose. The ratio differed from unity by more than the standard error of a single determination of the specific radioactivity of blood or plasma glucose except from 10 to 17min. after injection of [(14)C]glucose and from 22 to 30min. after injection of [(14)C]fructose. At all other times specific radioactivities in blood had to be corrected to give specific radioactivities in plasma. How to do so is described. 4. The P/B ratios were accounted for by a turnover of glucose in erythrocytes of 0.14mumole/min./ml. of erythrocytes. 5. Metabolism of glucose in rat erythrocytes is unlikely to be a major source of lactate.     

The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.     

A study on the precursors of the acetyl moiety of acetylcholine in brain slices. Observations on the compartmentalization of the acetyl-coenzyme A pool

1. A method was devised for the determination of the specific radioactivity of the acetyl moiety of acetylcholine synthesized from various (14)C-labelled substrates. 2. The precursor for the acetyl moiety of acetylcholine was studied in slices of striatum and cerebral cortex from rat and guinea-pig brain. Incorporation of radioactivity into acetylcholine was determined after incubating the slices in the presence of [2-(14)C]acetate, [(14)C]bicarbonate, [1,5-(14)C]citrate, dl-[1- or 5-(14)C]glutamate or [1- or 2-(14)C]pyruvate. 3. After incubation for 1h, acetylcholine was accumulated significantly in both striatum slices (4.1nmol/mg of protein) and cerebral-cortex slices (0.57nmol/mg of protein) from the rat. Final concentrations were about 11 and 5 times respectively the initial values. 4. With slices from rat striatum, rat cerebral cortex and guinea-pig cerebral cortex, the specific radioactivity of acetylcholine derived from [2-(14)C]pyruvate was very high, reaching approx. 30, 20 and 6% respectively of the initial specific radioactivity of added pyruvate in the medium. With the striatum slices this high value was reached after incubation for 15min. Incorporation of radioactivity from [2-(14)C]acetate was only 1.25, 5.3 and 19.7% of that from [2-(14)C]pyruvate in rat striatum, rat cerebral-cortex and guinea-pig cerebral-cortex slices respectively. A small but definite incorporation was found from [5-(14)C]glutamate. No incorporation was found from the other substrates. The findings suggest that pyruvate is the most important precursor for the synthesis of the acetyl moiety of acetylcholine in brain slices. 5. The specific radioactivity of acetylcholine relative to that of citrate when [2-(14)C]pyruvate was used compared with that obtained when [2-(14)C]acetate was used. A marked difference was found in all slices, suggesting metabolic compartmentation of the acetyl-CoA pool.     

Decrease of hepatic mono and oligo adenosine diphosphoribose content and augmentation of [14C]ribose incorporation during induction of growth by bovine growth hormone in hypophysectomized rats

Following $\text{in}\text{vivo}$ pulse labeling with [¹⁴C]ribose the specific radioactivities of mono- and polyadenosine diphosphoribose, NAD and adenine nucleotides were determined in livers of hypophysectomized Long Evans rats. These analyses were also performed after induction of growth with four successive daily injections of bovine growth hormone. As a consequence of brief treatment with growth hormone polyadenosine diphosphoribose content markedly diminished whereas its specific radioactivity increased. NAD concentration did not vary but its specific radioactivity increased similarly to that of the homopolymer. The steady state concentration of adenine nucleotides remained unchanged, except for ATP which decreased, and their specific radioactivities uniformly decreased.