Rapid communication: sequencing of the porcine agouti-related protein (AGRP) gene.
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Agouti-Related Protein↗
Biomedical subjects
T M Cheesbrough
Publications and source records attributed to T M Cheesbrough.
Warm growth temperatures decrease soybean cholinephosphotransferase activity.
The activity of cytidine 5'-diphosphate (CDP) choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) in developing soybean (Glycine max L. var Williams 82) seeds was 3 to 5 times higher in cotyledons grown at 20 degrees C than in those grown at 35 degrees C. Some characteristics of the enzyme from cotyledons cultured at 20 and 35 degrees C were compared. In preparations from both growth temperatures, the enzyme showed a pH optimum of 7, K(m) of 7.0 micromolar for CDP-choline, and an optimum assay temperature of 45 degrees C. Both enzyme preparations were stimulated by increasing concentrations of Mg(2+) or Mn(2+), up to 10 millimolar and 50 micromolar, respectively, though Mn(2+) produced lower activities than Mg(2+). Enzymes from both 20 and 35 degrees C show the same specificity for exogenous diacylglycerol. No metabolic effectors were detected by addition of heat treated extracts to the assay mixture. The above findings suggest that the higher enzyme activity at 20 degrees C can be attributed to a higher level of the enzyme rather than to the involvement of isozymes or metabolic effectors. Enzyme activity decreased rapidly during culture at 35 degrees C, indicating a rapid turnover of the enzyme. The level of temperature modulation was found to be a function of seed developmental stage.
Journal Article↗
Decreased growth temperature increases soybean stearoyl-acyl carrier protein desaturase activity.
Developing soybean (Glycine max) seeds respond to a change in growth temperature by changing the level of stearoyl acyl carrier protein desaturase activity in the tissue. After 20 hours in liquid culture, seeds grown at 20 degrees C show an increase in activity while seeds grown at 35 degrees C show a decrease in activity, relative to their preculture levels. Analysis of the enzyme from both growth conditions shows the change not to be due to induction of kinetically distinct iosenzymes; desaturase activities from both 20 degrees C and 35 degrees C have identical behavior with regard to pH, temperature optimum, substrate concentration and cofactor requirements. Experiments with boiled extracts indicate that the modulation is not caused by induction of metabolic effectors. From these data, it appears that stearoyl-acyl carrier protein desaturase responds to changes in growth temperature by altering the level of active enzyme present in the tissue. The magnitude of this response is a function of the developmental stage of the seed and not a function of the growth conditions of the parent plant. Changing the age of the seeds from early late R5 changed the ratio of 20:35 degrees C activity from 3.8:1 to 1.2:1. Changing the temperature at which the parent plants were grown over a range from 20/12 degrees C to 34/28 degrees C (day/night) produced only minor, and inconsistent, changes in the ratio of 20:35 degrees C activities.
Journal Article↗
Molecular cloning, nucleotide sequence, and tissue distribution of malonyl-CoA decarboxylase.
Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.
Amino Acid Sequence↗
Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature.
Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35 degrees C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20 degrees C as those cultured at 25 degrees C. Both desaturases had negligible activity in seeds cultured at 35 degrees C compared to seeds cultured at 20 degrees C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35 degrees C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20 degrees C cultures. Activities were twofold lower at 25 degrees C and a further threefold lower in 35 degrees C cultures. Cultures from 25 and 35 degrees C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20 degrees C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not.
Journal Article↗
Microsomal preparation from an animal tissue catalyzes release of carbon monoxide from a fatty aldehyde to generate an alkane.
Alkanes are widely distributed in nature and impaired alkane synthesis was implicated in certain neurological disorders. However, the mechanism of synthesis of alkanes in animals is unknown. Our search to find a convenient animal tissue to study alkane biosynthesis resulted in the finding that the uropygial gland (a modified sebaceous gland) of the eared grebe (Podiceps nigricollis) produces large amounts of alkanes. These alkanes, which constitute 35-41% of the total lipid produced, are mainly C21, C23, C25, and C27 n-alkanes. Cell free homogenates of this tissue synthesized alkanes from both fatty acid and aldehyde in the absence of O2. Differential centrifugation of the homogenates indicated that this activity was located in the microsomal fraction. With isolated microsomes conversion of fatty acid to alkane required CoA, ATP, and NADH whereas conversion of an aldehyde to alkane did not require the addition of cofactors. That the final step in alkane synthesis is a decarbonylation was shown by the stoichiometric production of heptadecane and CO from octadecanal. CO was identified by adsorption to RhCl [(C6H6)3P]3 and oxidation of the trapped CO to CO2 by watergas shift reaction. The enzyme preparation also catalyzed incorporation of 14C from 14CO into octadecanal showing the reversible nature of the decarbonylase. This decarbonylase had a sharp pH optimum at 7.0, a Kapp of 180 microM and a V1/2 of 90 rho mol/min/mg protein for octadecanal. The enzyme was inhibited by the metal chelators EDTA, O-phenanthroline, and 8-hydroxyquinoline, but not by KCN. It was stimulated nearly 3-fold by 5 microM 2-mercaptoethanol and inhibited by the presence of O2. During the conversion of [1-3H]octadecanal to heptadecane, 3H was lost to water and 3H from 3H2O was incorporated into the alkane generated from unlabeled octadecanal. The mechanism of the decarbonylation and the nature of the enzyme remain to be elucidated.
Aldehydes↗
Developmental pattern of the expression of malonyl-CoA decarboxylase gene and the production of unique lipids in the goose uropygial glands.
The abundant fatty acid synthase in the uropygial gland of goose generates multimethyl-branched fatty acids as the major product because of the unique presence of the cytoplasmic malonyl-CoA decarboxylase which assures that only methylmalonyl-CoA is available to the synthase. If this conclusion is valid, the developmental pattern of expression of the gene for this tissue-specific decarboxylase should correlate with the appearance of other lipogenic enzymes and the production of the unique lipids. To test this possibility the levels of the decarboxylase, acetyl-CoA carboxylase, and fatty acid synthase in the gland of the embryonic and neonatal goose were measured by immunodiffusion and immunoblot assays for the proteins as well as the enzyme assays for the catalytic activities. Malonyl-CoA decarboxylase appeared several days before hatching as did the other two lipogenic enzymes and reached half-maximal levels by hatching. The levels of expression of the malonyl-CoA decarboxylase gene and cytoplasmic actin gene, which is not expected to be developmentally regulated, were measured by dot-blot analysis using cloned cDNA for the two proteins. The decarboxylase transcripts appeared 4 days prior to hatching and reached maximal levels by hatching, whereas the levels of cytoplasmic actin gene transcripts showed very little change. The appearance of oil droplets in the glands was clearly seen soon after hatching. These results show that malonyl-CoA decarboxylase gene expression is developmentally regulated in a manner consistent with its proposed role in the synthesis of the unique lipids of the uropygial gland.
Aging↗
Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck.
In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.
Amino Acid Sequence↗
Purification and characterization of a fatty acyl-CoA hydrolase from the uropygial glands of Peking ducks (Anas domesticus).
In a previous communication the occurrence of a medium-chain acyl-CoA hydrolase designated thioesterase-B in the uropygial gland of mallard ducks was reported [L. Rogers, P. E. Kolattukudy, and M. J. de Renobales (1982) J. Biol. Chem. 257, 880-886]. In the present study, thioesterase-B was purified from the postmicrosomal supernatant of homogenized uropygial glands from Peking ducks (Anas domesticus). Most of the contaminating thioesterase activities were removed by ammonium sulfate fractionation. The 55% ammonium sulfate supernatant, containing thioesterase-B, was chromatographed on hydroxylapatite followed by gel filtration on Sephadex G-100. The remaining contaminants were removed by chromatofocusing followed by desalting on Sephadex G-75. This procedure gave a 26% yield with a nearly 200-fold purification. Gel filtration of the purified enzyme showed that the molecular weight of the native enzyme was 56,300, whereas sodium dodecyl sulfate-gel electrophoresis of components separated by chromatofocusing showed that the purified enzyme contained enzymatically active proteins of molecular weights 59,400, 58,300, 56,000, and 55,800. The four species differed slightly in pI (4.9, 4.7, 4.45, and 4.40) but they were kinetically and immunologically indistinguishable. All four had the same N-terminal sequence. The purified thioesterase preparation showed a pH optimum of 9.3 with C12-CoA but the pH optimum was dependent on the chain length of the acyl group. At pH 8.0, C10 was the preferred substrate with less activity on C12, C8, and C14. The enzymatic activity was stimulated by bovine serum albumin and was inhibited by p-hydroxymercuribenzoate. Involvement of active serine in catalysis was suggested by inhibition of the enzyme by diethylpyrocarbonate, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride.
Amino Acids↗
Alkane biosynthesis by decarbonylation of aldehydes catalyzed by a particulate preparation from Pisum sativum.
Mechanism of enzymatic conversion of a fatty acid to the corresponding alkane by the loss of the carboxyl carbon was investigated with particulate preparations from Pisum sativum. A heavy particulate preparation (sp. gr., 1.30 g/cm3) isolated by two density-gradient centrifugation steps catalyzed conversion of octadecanal to heptadecane and CO. Experiments with [1-3H,1-14C]octadecanal showed the stoichiometry of the reaction and retention of the aldehydic hydrogen in the alkane during this enzymatic decarbonylation. This decarbonylase showed an optimal pH of 7.0 and a Km of 35 microM for the aldehyde. This enzyme was severely inhibited by metal ion chelators and showed no requirement for any cofactors. Microsomal preparations and the particulate fractions from the first density-gradient step catalyzed acyl-CoA reduction to the corresponding aldehyde. Electron microscopic examination showed the presence of fragments of cell wall/cuticle but no vesicles in the decarbonylase preparation. It is concluded that the aldehydes produced by the acyl-CoA reductase located in the endomembranes of the epidermal cells are converted to alkanes by the decarbonylase located in the cell wall/cuticle region.
Aldehyde-Lyases↗
Transverse Distribution of Phospholipids in Organelle Membranes from Ricinus communis L. var. Hale Endosperm: MITOCHONDRIA AND GLYOXYSOMES.
Phospholipase A(2) (Naja naja), the nonpenetrating dye trinitrobenzene sulfonate, and the penetrating dye dinitrofluorobenzene, were used to determine the transmembrane distributions of phospholipids of mitochondria and glyoxysomes isolated from endosperm tissue of castor bean (Ricinus communis L. var. Hale). These studies indicated that the phospholipid distributions were distinctly asymmetric in the accessible (reacted with the probes without total membrane disruption by detergents) pools of the glyoxysomal and inner mitochondrial membranes, but more nearly symmetric in the outer mitochondrial membrane. However, significant quantities of the phospholipids of the mitochondrial membranes were inaccessible to the probes used. An increased accessibility of the phospholipids of all membranes following Triton X-100 dispersion was found, and protein to phospholipid ratios in organelle membranes were found to correlate inversely with the accessibility of the phospholipids to the probes. The inaccessible phospholipids may be involved in lipid-protein interactions.
Journal Article↗