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A Witkowski

Publications and source records attributed to A Witkowski.

32 records · Page 2Linked to original sources
PubMed1988

Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

Amino Acid Sequence↗
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

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

Pleiotropic effect of anticapsin on HeLa S3 cells.

Anticapsin, the terminal epoxyaminoacid moiety of tetaine, inhibits irreversibly growth of HeLa S3 cells. The antibiotic decreases to a similar extent incorporation of 3H-labelled precursors into nucleic acids and protein in intact cells: inhibition of protein synthesis prevails on prolonged incubation. Also incorporation of [3H]dTTP and [3H]UTP is inhibited in the presence of anticapsin into permeabilized cells. These effects, however, are not due to the interference with DNA or RNA polymerases since anticapsin only slightly suppresses RNA polymerase activity and has no effect on DNA polymerase in the cell-free systems. The results indicate that the mechanism of antiproliferative action of anticapsin in HeLa S3 cells differs from that of tetaine and imply that inhibition of protein synthesis might be the primary effect of anticapsin.

Alanine↗
PubMed1985

Differential inhibition of DNA and RNA biosynthesis in HeLa S3 cells by tetaine, a dipeptide antibiotic.

A dipeptide antibiotic, tetaine, was found to diminish the rate of incorporation of 3H-labelled precursors into nucleic acids of intact and permeabilized HeLa S3 cells with concomitant negligible effect on protein synthesis. Comparison of the inhibitory effects of tetaine indicates that the antibiotic at 0.03-0.1 mM is a selective inhibitor of cellular DNA biosynthesis and, at higher concentration, of DNA and RNA biosynthesis. Tetaine is also an inhibitor of DNA and RNA polymerase reactions in a cell-free system, as determined using partially purified extracts from HeLa S3 cells that served as a source of the enzymes. The pretreatment experiments showed that tetaine inactivated the polymerases without affecting DNA template function. The tetaine effect on biosynthesis of nucleic acids in HeLa S3 cells can be attributed rather to the intact antibiotic than to the product of its enzymatic cleavage, anticapsin.

Alanine↗
PubMed1985

Inhibition of the functional interaction between fatty acid synthetase and thioesterase II by modification of a single cysteine thiol on the thioesterase.

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete, monomeric, serine active-site enzyme, modifies the product specificity of the de novo lipogenic pathway by hydrolyzing the thioester bond linking the growing acyl chain to the 4'-phosphopantetheine of the fatty acid synthetase. The mechanism of interaction of thioesterase II and fatty acid synthetase has been studied by probing the thioesterase with sulfhydryl-modifying reagents. Modification of a single cysteine thiol with 5,5'-dithiobisnitrobenzoate destroyed the ability of thioesterase II to catalyze hydrolysis of S-acyl fatty acid synthetase thioesters but had no effect on the ability of the enzyme to hydrolyze the model substrate, decanoyl-S-pantetheine. The inhibition was readily reversed on removal of the thionitrobenzoate moiety from the thioesterase with dithiothreitol. The results of kinetic experiments indicated that loss of the capacity of the thioesterase to hydrolyze the natural substrate could be attributed to an inability of the 5,5'-dithiobisnitrobenzoate-modified enzyme to bind to the fatty acid synthetase. Modification of the same cysteine thiol with methyl methanethiolsulfonate did not affect the ability of thioesterase II to hydrolyze either the natural or model substrates. The results are interpreted to indicate that cysteine thiol, remote from the catalytic active-site serine residue, is present on a binding domain of the thioesterase which interfaces with the fatty acid synthetase. Modification of this thiol with the large thionitrobenzoate moiety, but not with the small CH3S- moiety, inhibits the functional interaction either by steric hindrance or by perturbation of the polypeptide configuration at the binding domain.

Animals↗
PubMed1984

Inhibition of the biosynthesis of deoxyribonucleic acid, ribonucleic acid and protein in HeLa S3 cells by cucurbitacins, glucocorticoid-like cytotoxic triterpenes.

Cucurbitacins were found to inhibit the incorporation of radioactive precursors into DNA, RNA and protein in intact and permeabilized HeLa S3 cells. The observed inhibition was rapid and irreversible although the maximal effect (almost complete inhibition) required several hours of cell exposition to the agent. The magnitude of the inhibition was, with some exceptions, nearly the same for all three precursors within the entire range of cucurbitacin concentrations examined. The ID50 values (concentrations required to produce half-maximal inhibition of the macromolecule biosynthesis) determined for several cucurbitacins were very close to their respective ED50 values (those for half-maximal inhibition of cell proliferation). Parallel with the inhibition of [3H]-labelled precursor incorporations into nucleic acids, cucurbitacin diminished the [3H]thymidine and [3H]uridine nucleotides' pool sizes of HeLa S3 cells. No effect of cucurbitacin on the [3H]leucine pool was observed. The studies presented prove that cucurbitacins inhibit the biosynthesis of DNA, RNA and protein in HeLa S3 cells, and that these inhibitory effects are closely related to the inhibition of HeLa S3 cell proliferation by cucurbitacins. The mechanism of the inhibition is unknown but the obtained results suggest that cucurbitacins act upon an unidentified target, which results in the inhibition of macromolecule biosynthesis. It was also found that these inhibitory effects of cucurbitacins are neither mediated by glucocorticoid receptors nor require replication, transcription or translation.

Antineoplastic Agents↗
PubMed1981

Binding of the cytotoxic and antitumor triterpenes, cucurbitacins, to glucocorticoid receptors of HeLa cells.

1. The binding of cucurbitacins to glucocorticoid receptors in HeLa cell-free systems and in intact cells was studied by competition with [3H]cortisol. Cucurbitacins were found to diminish the [3H]cortisol binding. 2. The difference in binding affinity at two temperatures suggest that cucurbitacins are metabolized under physiological conditions. 3. A linear correlation was observed between logarithms of relative binding affinities and of cytotoxic activities of cucurbitacins. Hence, the cucurbitacin binding to glucocorticoid receptors seems to be a necessary step for cytotoxic action of these compounds. 4. Cucurbitacin I was found to form cytoplasmic and nuclear salt-extractable complexes with glucocorticoid receptors of intact HeLa cells at 37 degrees C. Unlike cortisol, cucurbitacin I did not produce salt-resistant nuclear complexes.

Antineoplastic Agents↗