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S Smith

Publications and source records attributed to S Smith.

At least 793 records · Page 44Linked to original sources
PubMed1987

Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase.

Cloned cDNAs containing coding sequences for domains proximal to the carboxy terminus of the rat fatty acid synthetase have been isolated using an expression vector and domain-specific antibodies. The coding regions were assigned to specific domains of the multifunctional complex by identification of sequences coding for characterized peptide fragments and by recognition of sequences homologous to other monofunctional enzymes. Two clones contain the entire coding region for the acyl carrier protein domain. The sequence is flanked at the 3'-end by a region coding for the thioesterase domain and at the 5'-end by a sequence coding for a reductase, most likely the ketoreductase domain. Thus the ordering of these domain-coding regions in the fatty acid synthetase mRNA is established. The acyl carrier protein domain exhibits about 25% homology with that of the discrete monofunctional acyl carrier proteins of Escherichia coli, spinach and barley, the ketoreductase domain exhibits about 25% homology with bacterial dihydrofolate reductases and the active site of the thioesterase domain exhibits both primary and secondary structural features common to the serine proteases. These findings lend support to the hypothesis that the polyfunctional fatty acid synthetase probably arose by a complex evolutionary process involving fusion of genes coding for seven individual enzymes.

Acyl Carrier Protein↗
PubMed1987

Cloning and sequencing of the medium-chain S-acyl fatty acid synthetase thioester hydrolase cDNA from rat mammary gland.

cDNA clones coding for the medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II) from rat mammary gland were identified in a bacteriophage lambda gt11 library and their nucleotide sequences were determined. The predicted coding region spans 263 amino acid residues and includes a sequence identical with that of a peptide derived from the enzyme active site. The rat thioesterase II cDNA sequence exhibits homology with that of a thioesterase found in duck uropygial glands.

Amino Acid Sequence↗
PubMed1987

Inhibition of mammalian fatty acid synthetase activity by NADP involves decreased mobility of the 4'-phosphopantetheine prosthetic group.

Mammalian fatty acid synthetase carrying a 3-keto, 3-hydroxy, or 2-enoyl acyl-enzyme intermediate on the 4'-phosphopantetheine thiol is reversibly inhibited by binding of NADP to the enoyl reductase domain. Acyl moieties which can normally leave the enzyme by thioester hydrolysis or by transfer to a CoA acceptor cannot readily be removed from the NADP-inhibited enzyme; in addition, 3-keto or 2-enoyl moieties attached to the enzyme 4'-phosphopantetheine cannot readily be reduced when NADP is replaced by NADPH, even though model substrates can be reduced immediately. Reactivation of the NADP-inhibited 3-ketoacyl-enzyme, by exposure to NADPH, is paralleled by reduction and dehydration of the 3-ketoacyl moiety to a saturated acyl moiety without accumulation of either the 3-hydroxy or 2-enoyl acyl-enzyme intermediates, indicating that once the 4'-phosphopantetheine engages the ketoacyl moiety in the ketoreductase domain, subsequent reactions occur very rapidly. The results are consistent with a hypothesis which proposes that NADP binding to the enoyl reductase domain of fatty acid synthetase carrying an acyl intermediate other than a saturated moiety induces a conformational change in the enzyme that results in decreased mobility of the 4'-phosphopantetheine prosthetic group. Normal mobility of the prosthetic group, essential for transfer of acyl-enzyme intermediates through the active sites of the various functional domains, is restored relatively slowly when NADP is replaced by NADPH. It remains to be determined whether this modulation by pyridine nucleotides observed in vitro plays a role in the regulation of fatty acid synthetase activity in vivo.

Animals↗
PubMed1987

Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland.

The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated Mr of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis [Poulose, A.J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 15953-15958]. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.

Amino Acid Sequence↗
PubMed1987

A novel procedure for the preparation and characterization of catalytically active fatty acid synthetase immobilized on sepharose beads.

A novel procedure for immobilization of enzymatically active fatty acid synthetase is presented. The enzyme is coupled to a Sepharose 4B matrix containing covalently attached antibodies which recognize, and bind specifically to, the thioesterase domain of this polyfunctional enzyme. A continuous flow system is described for assay of the immobilized enzyme. Fatty acid synthetase activity apparently is not limited by movement of substrates through the Nernst diffusion layer surrounding the matrix particles, since normal Michaelis-Menten kinetics are observed and reaction rates are independent of flow rate. The Km values for acetyl-CoA and malonyl-CoA, the pH/activity profile, and the reaction products are essentially the same as for the freely soluble enzyme, although the specific activity is lower by about 55%. The preparation and characterization of immobilized subunits of the enzyme could provide a valuable approach for studying the role of structural and functional subunit interactions in the enzyme. In addition, the immobilized enzyme offers a model for studying the properties of this enzyme in a highly structured environment such as might exist in vivo, permitting study of both physical and functional interactions of fatty acid synthetase with other lipogenic enzymes.

Acetyl Coenzyme A↗
PubMed1987

Interaction of rat mammary gland thioesterase II with fatty acid synthetase is dependent on the presence of acyl chains on the synthetase.

The interaction between rat mammary gland thioesterase II and fatty acid synthetase has been studied by a variety of physicochemical techniques. Pyrene-labeled thioesterase II does not exhibit increased fluorescence anisotropy when mixed with fatty acid synthetase, suggesting that the enzymes do not readily form a complex. Nevertheless, the functional interaction between the enzymes can be easily demonstrated by observing the hydrolysis, by unmodified thioesterase II, of acyl chains from their thioester linkage to the 4-phosphopantetheine of the fatty acid synthetase. This hydrolytic reaction is not inhibited even in the presence of a large excess of fatty acid synthetase with vacant 4'-phosphopantetheine thiols, indicating that interaction occurs only between thioesterase and fatty acid synthetase species which carry acyl chains on the 4'-phosphopantetheine thiols. A novel model system was devised which allowed us to explore the nature of the physical interaction between the two enzymes under conditions where the synthetase was actively engaged in acyl chain assembly. Fatty acid synthetase was treated with phenylmethanesulfonyl fluoride to inhibit its resident thioesterase activity, immobilized via a specific antibody to a column of Sepharose 4B, and exposed to the substrates required for acyl-enzyme assembly. When thioesterase II was introduced to the column, it passed through unretarded even though it efficiently catalyzed hydrolysis of the immobilized S-acyl synthetase en route. These results indicate that the two enzymes associate when an acyl chain is present on the synthetase and that they dissociate rapidly following completion of the catalytic process. Thus, the mammary system differs from that of the avian uropygial gland in which the two enzymes associate to form a stable complex even in the absence of substrates.

Animals↗
PubMed1987

Amino acid sequence of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland.

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57-residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3-14C]diisopropyl-fluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active-site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a lambda gt11 expression library. The active center contains the motif Gly-Xaa-Ser-Xaa-Gly, characteristic of the serine esterase family of enzymes. A seven-residue region around the essential serine of the rat mammary thioesterase II, Phe-Gly-Met-Ser-Phe-Gly-Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57-amino-acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.

Amino Acid Sequence↗
PubMed1987

Ultrastructure of the final nuclear maturation of bovine oocytes in vitro.

Cumulus-oocyte complexes were obtained from cow ovaries by aspiration from small (1-6 mm in diameter) antral follicles after slaughter. Complexes with a compact multilayered cumulus investment were cultured and subsequently processed for electron microscopy after various periods of culture. By morphological criteria the oocytes could be divided into the following sequence of meiotic stages. The oocyte nucleus I stage was characterized by a spherical nucleus located peripherally in the ooplasm while undulation of the nuclear envelope and initial chromatin condensation was seen at the oocyte nucleus II stage. The oocyte nucleus breakdown stage was characterized by formation of long slender projections from the nuclear envelope in which the envelope doubled back on itself, appearance of dense areas and haphazardly oriented microtubules in the nucleus, marked condensation of the chromatin, and dissolution of the nuclear envelope into irregular vesicles and tubules. The condensed chromatin I stage was characterized by the location of condensed chromatin configuration and uniformly oriented microtubules in a dense area peripherally in the ooplasm while the final condensed chromatin II stage was characterized by a gradual invasion of condensed chromatin configurations into a dense area combined with the presence of the first polar body in the perivitelline space.

Animals↗
PubMed1987

Accessibility of case and gender contrasts for agent--object assignment in Broca's aphasics and fluent anomics.

Yugoslav agrammatic Broca's aphasics, fluent anomics, and control subjects were tested for comprehension of agent-object relations in a series of simple Serbo-Croatian sentences in the conversational past tense, consisting of two nouns and a transitive action verb. The availability of two closed class cues--case contrasts and gender contrasts--as well as the availability of an open class lexical cue--animacy contrasts--was varied across sentences. An analysis of subjects' agent-object assignments yielded the following results: Both Broca's aphasics and fluent anomics showed a selective impairment in sensitivity to closed class morphology, although anomics were considerably less impaired than Broca's aphasics. This finding was interpreted as evidence for a non-syndrome-dependent vulnerability of abilities to process closed class morphology for comprehension. In addition, the pattern of agent-object assignments for Broca's aphasics revealed that the degree to which they were able to access the two closed class cues depended on a convergence of the various cues to agent-object relations. In particular, a convergence of case and gender contrasts had an interactive effect on Broca's responses: Gender agreement cues were accessed only in the presence of convergent case contrasts. Conversely, sensitivity to case contrasts was dramatically heightened in the presence of convergent gender contrasts. These results were consistent with a view that the two cues function as intrasentential "primes" or "retrieval cues" for one another.

Adult↗
PubMed1987

The correlation between surface immunoglobulin expression and the leucocyte-common antigen in B-cell chronic lymphocytic leukaemia.

The expression of the 230 kD glycoprotein of the leucocyte-common antigen by the leukaemic lymphocytes of patients with B-cell CLL, PLL and HCL and of normal B lymphocytes has been measured in a flow cytometer by the binding of the McAb F8-11-13 and surface tritiation. Its expression has been correlated with the expression of surface immunoglobulin heavy chain and light chain. The levels of binding of F8-11-13 and sIg cover a wide range within the CLL panel, ranging from very low (10% normal) to levels found in the other two leukaemias and normal cells. The progressive deviation from normal in the CLL panel is accompanied in all but a minority of patients by the expression of the leucocyte sialoglycoprotein.

B-Lymphocytes↗
PubMed1987

Transgenic mice with increased Cu/Zn-superoxide dismutase activity: animal model of dosage effects in Down syndrome.

Down syndrome, the phenotypic expression of human trisomy 21, is presumed to result from a 1.5-fold increase in the expression of the genes on human chromosome 21. As an approach to the development of an animal model for Down syndrome, several strains of transgenic mice that carry the human Cu/Zn-superoxide dismutase gene have been prepared. These animals express the transgene in a manner similar to that of humans, with 0.9- and 0.7-kilobase transcripts in a 1:4 ratio, and synthesize the human enzyme in an active form capable of forming human-mouse enzyme heterodimers. Cu/Zn-superoxide superoxide dismutase activity is increased from 1.6- to 6.0-fold in the brains of four transgenic strains and to an equal or lesser extent in several other tissues. These animals provide a unique system for studying the consequences of increased dosage of the Cu/Zn-superoxide dismutase gene in Down syndrome and the role of this enzyme in a variety of other pathological processes.

Alzheimer Disease↗
PubMed1987

Multiple forms of basic fibroblast growth factor: amino-terminal cleavages by tumor cell- and brain cell-derived acid proteinases.

Basic fibroblast growth factor (FGF) was purified by heparin-Sepharose chromatography from two sources, brain and hepatoma cells. Brain cell-derived basic FGF (brFGF) and hepatoma cell-derived basic FGF (heFGF) were found to exist in multiple forms whose molecular weights depended on whether they were extracted from their respective tissue or cells at neutral or acid pH. When extracted at pH 7.0 brFGF and heFGF comigrated on NaDodSO4/PAGE with a Mr of approximately 18,400. When extracted at pHs 3.5-4.5, acid proteinases cleaved brFGF and heFGF to lower molecular weight forms but to different extents. brFGF was cleaved to a Mr 18,000 form at acid pH by a brain-derived acid proteinase that could be inhibited by pepstatin. heFGF was cleaved mostly to a Mr 16,500 form at acid pH by a hepatoma cell-derived acid proteinase that was inhibited by leupeptin. Electrophoretic transfer blot analysis using site-specific anti-FGF antibodies suggested that the cleavages occurred at the amino-terminal ends of brFGF and heFGF. Cleavage to lower molecular weight forms of brFGF and heFGF did not affect growth factor activity or chromatographic behavior on heparin-Sepharose columns.

Amino Acid Sequence↗
PubMed1987

Involvement of nucleophiles in the inactivation of atracurium.

Atracurium was incubated in 0.9% sodium chloride solution at pH 7.4 and 37 degrees C. The following compounds were added to the incubation solution: propionic acid, alanine and cysteine (0.1 mol litre-1 final concentration). Metocurine was also incubated with cysteine under identical conditions. Aliquots of the incubation solutions were injected i.v. to anaesthetized rats at the start (0 min) and at the end of incubation (45 min), and the indirectly elicited, single twitches of the gastrocnemius muscle were observed. Whereas inactivation of atracurium proceeded slowly in the control solution and in the presence of sodium propionate, the presence of alanine and, especially, of cysteine markedly enhanced the inactivation. Incubation of metocurine with cysteine did not alter the pharmacological responses. The concentration of cysteine decreased progressively during its incubation with atracurium (37 degrees C, pH 7.4). We conclude that the data are compatible with the postulate that a nucleophile may enhance the degradation of atracurium by a reaction with the parent compound (nucleophilic substitution reaction), by reaction with acrylates (Michael addition) or both.

Alanine↗