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J K Stoops

Publications and source records attributed to J K Stoops.

At least 55 records · Page 3Linked to original sources
PubMed1980

Yeast fatty acid synthetase: structure-function relationship and nature of the beta-ketoacyl synthetase site.

Yeast fatty acid synthetase consists of two multifunctional proteins, alpha and beta, which are arranged in a complex of alpha(6)beta(6). Electron microscopic studies of this complex led to a model for the synthetase as an ovate structure consisting of an equatorial plate-like structure to which six arches are equally distributed on either side. The bifunctional reagent 1,3-dibromo-2-propanone inhibits the synthetase by reacting rapidly (t((1/2)) approximately 7 sec) with two juxtapositioned active sulfhydryl groups. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the dibromopropanone-inhibited synthetase shows that the beta subunit is intact and the alpha subunit nearly absent with a concomitant appearance of oligomers with an estimated molecular weight of 0.4-1.2 x 10(6). These results indicate that the alpha subunits are crosslinked by this bifunctional reagent. Because the active centers of dibromopropanone are 5 A apart, it is concluded that the alpha subunits are closely packed so that the reacting thiols of the adjacent alpha subunits are within 5 A of each other. Furthermore, because the plate-like structures in our model are the only components that are arranged closely enough to satisfy this requirement, it is proposed that the alpha subunits are the "plates" and the beta subunits therefore are the "arches." Assay of the partial reactions shows that dibromopropanone inhibits the beta-ketoacyl synthetase reaction but none of the six other partial reactions, indicating that the site of action of the bifunctional reagent is the condensing reaction. This conclusion was supported by the finding that pretreatment of the synthetase with acetyl-CoA or iodoacetamide prevented dibromopropanone from interacting at this site and obviated the formation of the crosslinked oligomer. These observations and other lead us to propose that a site of action of the dibromopropanone is the active cysteine-SH of the beta-ketoacyl synthetase of one alpha subunit and the pantetheine-SH of the acyl carrier protein moiety of an adjacent alpha subunit. Thus, the enzymically active center of the beta-ketoacyl synthetase consists of an acyl group attached to the cysteine-SH of one alpha subunit (plate) and a malonyl group attached to the pantetheine-SH of an adjacent alpha subunit. This arrangement appears to be necessary for the coupling of the acyl and beta-carbon of the malonyl group to occur to yield CO(2) and the beta-ketoacyl product.

Alkylating Agents↗
PubMed1979

Effect of estrogen on fatty acid synthetase in the chicken oviduct and liver.

Estrogen administered to one-month-old female chickens resulted in a 180-fold increase in the amount of fatty acid synthetase, a seven-fold increase in the enzyme activity per gram of tissue and a 25-fold increase in the weight of the oviduct. In contrast, the fatty acid synthetase content in liver increased three-fold; activity per gram of tissue increased two-fold and the weight increased two-fold. The large increase in the fatty acid synthetase activity in the oviduct was due to a corresponding increase in the amount of the fatty acid synthetase protein since the specific activities of highly purified preparations of oviduct and liver fatty acid synthetases were the same and the two enzymes had the same end point as determined by immunoprecipitation. That the increase in activity of the oviduct enzyme is not due to a modification was further supported by physicochemical comparison of the oviduct enzyme with the chicken liver enzyme. Thus, the synthetase complexes have similar size, their subunit composition and size appear to be the same, and both are multifunctional enzymes. Finally, kinetic studies and product analyses indicated no catalytic difference between the enzyme induced by estrogen in the oviduct and the liver enzyme.

Adipose Tissue↗
PubMed1976

On the 4'-phosphopantetheine content of chicken and rat liver fatty acid synthetases.

The finding that animal synthetases are complexes consisting of two polypeptide chains (Stoops, J.K., Arslanian, M.J., Oh, Y.H., Vanaman, T.C., and Wakil, S.J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1940-1944) led us to investigate their 4'-phosphopantetheine content. We have found that the chicken and rat synthetases contain 1.6 to 2.2 mol of 4'-phosphopantetheine per mol of the complex. The implications of this finding concerning the structure of the complex and the biosynthetic pathway of fatty acid synthesis are discussed.

Alanine↗
PubMed1975

Organophosphate inhibitors: the reactions of bis(p-nitrophenyl) methyl phosphate with liver carboxylesterases and alpha-chymotrypsin.

Bis(p-nitrophenyl) methyl phosphate (BNMP) has been tested as a spectrophotometric titrant for a group of serine hydrolases. Bis(p-nitrophenyl) methyl phosphate reacts rapidly with liver carboxylesterases from chicken, sheep, and horse, and more slowly with alpha-chymotrypsin, releasing 2 mol of p-nitrophenol per active site titrated, and producing a phosphorylated enzyme very stable to dephosphorylation. However, pig liver carboxylesterase produces 2.2 mol of p-nitrophenol per active site titratedmreaction of pig and chicken liver carboxylesterases with bis(p-nitrophenyl) [3H]methyl [32P]phosphate clarified this differencemone molecule of the chicken enzyme reacts with one molecule of bis(p-nitrophenyl) methyl phosphate, releasing both p-nitrophenol residues, and resulting in an inhibited enzyme with one phosphorus atom and one methyl group covalently bound. Pig enzyme reacts rapidly, forming (presumably) methyl p-nitrophenyl phosphoryl-carboxylesterasemthis further reacts, concurrently producing methyl phosphoryl-carboxylesterase plus p-nitrophenol, or free enzyme plus methyl p-nitrophenyl phosphate, in the ratio of about 5 : 1 at pH 7.55. The free enzyme produced undergoes further reaction with bis(p-nitrophenyl) methyl phosphate until all the carboxylesterase is inhibited.

Animals↗
PubMed1975

Presence of two polypeptide chains comprising fatty acid synthetase.

Highly purified fatty acid synthetases of chicken and rat livers have molecular weights of 500,000 and dissociate in solutions of low ionic strength into subunits of molecular weight 250,000 with loss of synthetase activity. The subunits can be reassociated in phosphate buffer with full restoration of the activity. In the presence of sodium dodecyl sulfate or guanifine-HCl, the synthetases dissociate into polypeptide chains of molecular weight 220,000 as determined by sodium dodecyl sulfate-gel electrophoresis and sedimentation equilibrium. The polypeptide contains the 4-phosphopantetheine group and the [14C]acetyl and [4C]malonyl groups if the synthetases were prelabeled with [14C]acetyl-CoA and [14C]malonyl-CoA. Similar results were obtained with the synthetase from yeast, except the subunit has a molecular weight of 200,000. These observations indicate that the multi-catalytic activities of the synthetases and the acyl carrier protein are associated only with the two polypeptide chains. The findings suggest a novel structural organization for multienzyme complexes.

Acetates↗
PubMed1975

Carboxylesterases (EC 3.1.1). A comparison of some kinetic properties of horse, sheep, chicken, pig, and ox liver carboxylesterases.

A comparative study of the kinetic be,avior of horse, sheep, chicken, pig, and ox liver carboxylesterases is reported. The enzymes exhibit similar specificites towards a series of phenyl esters in which the acyl group is varied, and towards a series of butyrate esters in which the alcohol group is varied. Non-Michaelis-Menten kinetics are exhibited by the horse enzyme in the hydrolysis of methyl and ethyl butyrates, and by the pig enzyme with ethyl butyrate. Each enzyme exhibits inhibition by one or more substrates. A simple scheme which accounts for both activation and inhibition is discussed. pH-k(cat) profiles for the horse and chicken liver carboxylesterase-catalyzed hydrolyses of phenyl butyrate demonstrate dependencies on pK(a)S of 4.75 and 5.0, respectively.

Animals↗
PubMedDate not supplied

The novel three-dimensional structure of native human alpha 2-macroglobulin and comparisons with the structure of the methylamine derivative.

A three-dimensional reconstruction of alpha 2-macroglobulin (alpha 2 M) was computed from stain images. The structure appears to have point group symmetry 222 and, as also revealed by a tilt experiment, has the gross shape of a oval that displays a approximately 90 degrees twist in the body of the molecule. The reconstruction reveals a novel structure that consists of two Z-shaped components arranged in opposite orientation. These shapes are interconnected by two bridges at the elbow bends of the Z and by two archlike features that join their ends. The molecule has dimensions of approximately 190 x 125 x 120 A that encloses a 90 degrees twisted ellipsoidal shaped central cavity of 70 x 35 A. The cavity has four small openings arranged in a staggered configuration that extend to the outside. Serial slices of alpha 2 M and alpha 2 M-methylamine show that the bodies of the structures appear to be twisted in the opposite orientation. It is proposed that the four thioester bonds in the native molecule are responsible for maintaining its twisted configuration and that their cleavage with methylamine results in the structure becoming twisted in the opposite orientation. A comparison of average images derived from unstained particles of monoclonal Fab-labeled alpha 2 M and alpha 2 M-methylamine is consistent with this proposal. This unusual change in the handedness of alpha 2 M may have an important role in the encapsulation of the proteinase.

Binding Sites↗
PubMedDate not supplied

Three-dimensional structures of the human alpha 2-macroglobulin-methylamine and chymotrypsin complexes.

The three-dimensional structures of chymotrypsin- and methylamine-treated negatively stained human alpha 2-macroglobulin have been determined by weighted back projection from electron microscope data. Projections of the reconstructions show good concordance with two-dimensional averages of both stained and frozen-hydrated molecules. The reconstructions reveal that the H-shaped front projection of the molecule is related to the smaller ellipsoidal end view by a rotation of 90 degrees about the crossbar (minor axis) of the H. This finding is in agreement with tilt studies. The reconstruction of the alpha 2-macroglobulin-methylamine reveals an hour-glass shaped void which is filled by the two proteinase molecules in the reconstruction of alpha 2-macroglobulin-chymotrypsin. Protein plugs which appear to block the exterior entrances to the cavity may function to prevent access of proteins to the encapsulated proteinase and serve to block its escape. Extensive thresholding of each reconstruction leaves a "backbone" consisting of two side-by-side rod-like structures, suggesting that this is the arrangement of the two protomeric units which form the molecule. Both structures show some departure from the expected symmetry. The asymmetries are robust features of the reconstructions and may reflect structurally asymmetric features of the transformation from the native to the chymotrypsin-treated form of the molecule.

Binding Sites↗