I.L. Chaikoff

Factors influencing the pattern of fatty acids synthesized by cell-free preparations of lactating rat mammary gland

Abstract 1. 1. The pattern of fatty acids synthesized from [1- 14 C]acetate, [1- 14 C]acetyl-CoA, [1,5- 14 C 2 ]citrate and [l,3- 14 C 2 ]malonyl-CoA by various homogenate preparations of mammary glands of lactating rats was studied with the aid of gas-liquid chromatography. 2. 2. Addition of microsomes to the 100000× g supernatant fraction significantly decreased the incorporation of labeled acetate into C 10 and C 12 fatty acids and increased that into C 16 and C 18 acids. 3. 3. When [ 14 C]acetate or acetyl-CoA were the substrates in experiments with the 100000× g supernatant fraction, the isotope was incorporated predominantly into C 10 fatty acid. The labeled malonyl-CoA was predominantly converted to C 16 fatty acid. The pattern of incorporation of the 14 C of [1,5- 14 C 2 ]citrate was intermediate between these two. 4. 4. When an ammonium sulfate fraction of the 100000× g supernatant fluid, precipitating between 0 and 40% saturation, was used as the source of acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase, the pattern of conversion of all labeled substrates to fatty acids was the same, i.e., predominautely to C 16 acids. Addition of fractions precipitating at higher satuiations of ammonium sulfate had no effect on the pattern of conversion. 5. 5. When the ammonium sulfate fraction (0–29% saturation) rich in acetyl-CoA carboxylase was added to incubation mixtures containing a constant amount of a fraction (29–40% saturation) rich in fatty acid synthetase, the average chain length of the fatty acids synthesized from acetyl-CoA was decreased as the concentration of acetyl-CoA carboxylase was decreased, but the chain length of the fatty acids formed from the labeled malonyl-CoA was unchanged. 6. 6. The results indicate that the concentration of malonyl-CoA is a factor in determining the pattern of fatty acids formed by mammary gland. The mechanism for expression of this influence is discussed, along with other factors that might affect the pattern of fatty acids synthesized.

Esterification of cholesterol by rat adrenal gland homogenates and subcellular components.

Abstract This study demonstrates that the rat adrenal contains two enzymes that esterify cholesterol: one present principally in the mitochondria-rich fraction, the other most active in the microsome-rich fraction. The enzyme in the microsome-rich fraction had an optimum activity at pH 6.6 and required the addition of ATP, CoA, Mg 2+ and GSH for its activity. The enzyme in the mitochondria-rich fraction had an optimum activity at pH 5.0 and did not require the addition of these cofactors, but required cell sap or a lipid extract of cell sap for its activity. At least one of the ways in which cell sap exerted this stimulatory effect was by providing both free cholesterol and the fatty acid for the esterification. Both triolein and oleic acid served as the source of fatty acid for the esterification by the enzyme associated with the mitochondria-rich fraction, but the triolein was apparently hydrolyzed to free fatty acids before incorporation into the esters. Neither 2,4-dinitrophenol nor sodium arsenate inhibited the esterification, thereby suggesting that ATP was not involved in the activation of fatty acids before the esterification of cholesterol by that fraction of the rat adrenal gland.

Effects in vitro of thyrotropic hormone III. On incorporation of 1311 into a thyroglobulin-like protein by monolayer cultures of isolated sheep thyroid gland cells

Abstract 1. 1. Isolated sheep thyroid cells cultured in monolayers with 131I are capable of incorporating a considerable proportion of the isotope into a protein that has an electrophoretic mobility identical to that of purified thyroglobulin. In 1-day old cultures approx. 12–16% of the total 1311 was recovered in this protein. 2. 2. The 131I recovered in the electrophoretically separated thyroglobulin prepared from control cultures decreased sharply from the first to the third day, and thereafter continued to decrease (though less steeply) to the seventh day. 3. 3. Addition of thyrotropic hormone at the start of culturing enhanced the incorporation of 131I into thyroglobulin from the third to the seventh day. In 5-day-old cultures the 131I recovered as thyroglobulin in thyroid-stimulating-hormone-treated cells was about five times those of the controls. 4. 4. The effect of thyroid-stimulating hormone appeared to be specific for incorporation of the 131I into thyroglobulin; the incorporation of 131I into non-thyroglobulin proteins was not significantly affected. 5. 5. Addition of adrenocorticotropic hormone, luteinizing hormone, follicle-stimulating hormone and insulin had no effect on the incorporation of 131I into thyroglobulin by the monolayer cultures of thyroid cells. 6. 6. The mitochondrial and microsomal fractions prepared from the isolated cells cultured as monolayers contained an appreciable amount of protein bound 131I. Electrophoresis revealed that in both fractions, much of the 131I-labeled protein remained at the origin. 131I-labeled protein of the soluble fraction behaved like thyroglobulin as judged by ( I ) electrophoretic behaviour, (2) gel filtration on Sephadex G-200 and (3) reaction to anti-sheep-thyroglobulin antibodies. 7. 7. Mitochondria and microsomes contained significant amounts of free thyroxine. Peptide-bound thyroxine was present only in the protein of the soluble fraction. 8. 8. The relative roles of colloid and of cells in the biogenesis of thyroglobulin in the thyroid follicle are discussed.

The effect of nutritional state on lipoprotein lipase activity in isolated rat adipose tissue cells

Abstract Lipoprotein lipase was studied in adipose tissue cells isolated by collagenase treatment of parametrial fat pads obtained from rats fasted 3 days or rats fasted 3 days and refed I day. Lipoprotein lipase was associated with fat cells but not with stromal-vascular cells. Refeeding brought about an increase in the level of lipoprotein lipase in the isolated fat cells. Fat cells obtained from refed rats responded to heparin added in vitro by release of lipoprotein lipase into the incubating media. No such effect of heparin was observed with fat cells of fasted rats or with stromal-vascular cells of rats of either nutritional state. The addition of collagenase to the lipoprotein lipase assay system inhibited the production of free fatty acid. These results demonstrate that in adipose tissue the major site of occurrence and regulation of lipoprotein lipase activity is the fat cell. The possibility that the enzyme acts at or within the capillary wall cannot be excluded since any enzyme present outside the adipocyte itself might have been rendered inactive by the collagenase preparation during the process of cell isolation

Biosynthesis of thyroidal glycoprotein I. Uptake and incorporation of d-[1-14C]glucosamine into proteins of sheep thyroid gland slices and isolated cells

Abstract 1. 1. Sheep thyroid gland slices and isolated cells were shown to remove rapidly d -[1-14C]glucosamine from the incubation medium. The radioactivity appears first as trichloroacetic acid-soluble materials. This was followed by a conversion to trichloroacetic acid-insoluble substances. At least 30% of the added label could be recovered in both preparations, 20% as trichloroacetic acid-soluble derivatives and the remaining 10% as protein-bound materials. 2. 2. Chemical decarboxylation of d -[1-14C]glucosamine isolated from trichloroacetic acid-soluble materials of medium, trichloroacetic acid-soluble metabolites of slices and isolated cells, and hydrolysates of proteins showed that the radioactivity still resided mainly in C-1, indicating insignificant breaking of the hexose chain. 3. 3. The microsomes prepared from the slices contained the highest specific activity of the subcellular fractions indicating this particulate material as the most probable site of glycoprotein synthesis. 4. 4. d -[1-14C]Glucosamine was incorporated into a glycoprotein that closely resembled thyroglobulin in electrophoretic mobility on cellulose acetate paper strips and by chromatographic behavioron Sephadex G-200. 5. 5. It is suggested that the glucosamine is introduced to the peptide chains of the glycoproteins at some reaction steps before these proteins are released from the thyroid gland particulate fraction.

Evidence for the presence of 17-hydroxypregnenedione isomerase in beef adrenal cortex

Abstract 1. 1. Since 17α-hydroxypregnenolone is a precursor in the biosynthesis of corticosteroids, an attempt was made to determine whether 17α-hydroxypregn-5-ene-3,20-dione was isomerized by the same enzyme as was pregn-5-ene-3,20-dione or whether a separate enzyme, 17α-hydroxypregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) isomerase, also existed in the adrenal gland. 2. 2. Several fractions prepared from bovine adrenal cortex were assayed for 17α-hydroxypregn-5-ene-3,20-dione Delta; 5(6) −Δ 4(5) -isomerase and pregn-5-ene-3,20-dione gD 5(6) −Δ 4(5) -isomerase activities. Some fractions were free of 17α-hydroxypregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase activity, yet contained pregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase activity. From this and other findings it is inferred that 17α-hydroxypregn-5-ene-3,20-dione and pregn-5-ene-3,20-dione were isomerized by two distinct enzymes in the adrenal cortex. 3. 3. A scheme is presented indicating the different roles of 17α-hydroxypregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase, pregn-5-ene-3,20-dione Δ 5(6) −Δ 4(5) -isomerase, and androst-5-ene-3,17-dione Δ 5(6) −Δ 4(5) -isomerase in the biosynthesis of cortisol and aldosterone and in the metabolism of adrenal androgens.

The effect of insulin on iodine metabolism of fetal thyroid glands in organ culture

Abstract 1. 1. Thyroid glands excised from the fetuses of 21-day pregnant rats were cultured for four days in four different media: a basal medium; the basal medium to which insulin alone was added; the basal medium to whch thyrotropic hormone (TSH) alone was added; and the basal medium to which both TSH and insulin were added. 2. 2. Addition of insulin to the basal medium enhanced significantly the incorporation of 131 I into protein-bound monoiodotyrosine, diiodotyrosine and thyrosine. 3. 3. Addition of TSH to the basal medium, as reported earlier for thyroid glands removed from younger fetuses (16–16.5 and 17–17.5 days of pregnancy), had a pronounced effect on the functional activity of the embryonic thyroid glands explanted into culture. The addition of insulin to the TSH-containing medium further enhanced the 131 I utilization by the embryonic thyroid tissue, the formations of diiodotyrosine and thyroxine being particularly affected. 4. 4. The significance of this action of insulin is discussed.

Oxidation of reduced nicotinamide-adenine dinucleotide phosphate by soluble rat muscle α-glycerophosphate dehydrogenase. A comparison with purified lactate dehydrogenase and malate dehydrogenase

1. 1. NADPH was observed to disappear from a Tris-histidine medium (pH 7·5) in the presence of glucose 6-phosphate, ATP, magnesium ion and a particle-free supernatant fraction obtained from a rat-muscle homogenate. This phenomenon can be explained by direct oxidation of the NADPH by the soluble α-glycerohosphate dehydrogenase (l-glycerol-3-phosphate:NAD+ oxidoreductase, EC 1.1.18). From measurements of α-glycerophosphate dehydrogenase activity in the muscle preparation in the presence of NADPH, we can conclude that the capacity of this dehydrogenase to oxidize NADPH exceeds by thr the capacities of the dehydrogenases that act on glucose 6-phosphate and 6-phosphogluconate to generate NADPH. 2. 2. Kinetic characteristics of a α-glycerophosphate dehydrogenase of the particle-free fraction obtained from rat muscle and commercially purified (a) rabbit-muscle α-glycerophosphate dehydrogenase, (b) rabbit-muscle lactate dehydrogenase (l-lactate:NAD+ oxidoreductase, EC 1.1.1.27), and (c) porcine-heart malate dehydrogenase (l-malate:NAD+ oxidoreductase, EC 1.1.1.37) were investigated and found to be quite similar: for each enzyme, pH-optima were higher for the reaction with NADH than for that with NADPH; the substrate inhibited the enzymes when NADH acted as the coenzyme (especially at low pHs), but not when NADPH did; and the apparent Km values for NADH were considerably lower than those for NADPH. 3. 3. Inhibition of rabbit-muscle α-glycerophosphate dehydrogenase by NAD+ was observed with NADPH as the coenzyme, but not with NADH, NADP+ produced no inhibition with either coenzyme.

Amino acid incorporation into protein by cell-free preparations of sheep thyroid glands: The microsomal system

Abstract Some characteristics of the incorporation of amino acid carbon into protein by microsomal preparations of sheep thyroid glands are described. Amino acid incorporation was dependent on an ATP-generating system, guanosine triphosphate, glutathione, cell sap (100 000 × g supernatant) and Mg 2+ and was inhibited by puromycin. Chloramphenicol inhibited the incorporation of 14 C of [ 14 C]leucine but had no effect on that of phenylalanine carbon. Amino acid incorporation into protein by sheep thyroid microsomes was not sensitive to actinomycin D or deoxyribonuclease (EC 3.1.4.5). Because actinomycin D did not inhibit the incorporation of amino acid carbon into protein, the presence of a stable messenger RNA in the microsomal preparations of the sheep thyroid gland is suggested.

Biosynthesis of thyroidal glycoproteins: II. Incorporation of D-[1-14C]glucosamine into macromolecules by isolated thyroid cells. Effects of thyroid-stimulating hormone and puromycin

Abstract 1. 1. Isolated cells from sheep thyroid gland were shown to incorporate D -[1-14C]-glucosamine into glycoproteins of the subcellular fractions. The microsomal fraction was the most highly labeled. 2. 2. Puromycin caused 58–89% inhibition of D -[1-14C]glucosamine and 92–99% of L -[14C]lysine incorporation into the acid-insoluble materials of the whole cells. 3. 3. Thyroid stimulating hormone added in vitro resulted in an increased incorporation of either glucosamine (60–80%) or lysine (87–110%) into macromolecules of the isolated cells. 4. 4. When the isolated cells were exposed to either labeled glucosamine or lysine and then transferred to an unlabeled medium containing the relevant precursors, the specific activity of the cellular proteins as a whole decreased. Moreover, there was a concomitant increase in the acid-soluble radioactivity of the incubation medium.