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At least 1,621 records · Page 90Linked to original sources
PubMed1981

Immunochemical studies on catabolite inactivation of phosphoenolpyruvate carboxykinase in Saccharomyces cerevisiae.

Phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was purified to homogeneity. The enzyme is composed of four subunits of Mr = 64,000. Specific antibodies against phosphoenolpyruvate carboxykinase were raised in rabbits and purified by affinity chromatography. Phosphoenolpyruvate carboxykinase is rapidly inactivated when glucose is added to cells starved for carbon (Haarasilta, S., and Oura, E. (1975) Eur. J. Biochem. 52, 1-7; Gancedo, C., and Schwerzmann, K. (1976)( ARch. Microbiol. 109, 221-225). In the present study this inactivation has been analyzed by immunochemical techniques. It was found that the loss of catalytic activity is paralleled by a decrease in cross-reacting material which suggests degradation of the enzyme. In the absence of glucose the enzyme is degraded very slowly, which indicates that glucose-induced inactivation cannot simply be due to repression of enzyme synthesis in the presence of a rapid rate of degradation. Experiments with a proteinase-deficient mutant showed that proteinase B, carboxypeptidase Y, and carboxypeptidase S are not involved in the inactivation system.

Antibodies↗
PubMed1998

Human substance P receptor expressed in Chinese hamster ovary cells directly activates G(alpha q/11), G(alpha s), G(alpha o).

Substance P receptor (SPR) stably expressed in Chinese hamster ovary (CHO) cells stimulates at least three second messenger systems including phosphoinositide hydrolysis, cyclic AMP (cAMP) formation, and arachidonic acid release. Whether these second messenger systems are activated via single or multiple G proteins is not known. Therefore, in the present study we examined whether human SPR (hSPR) stably expressed in CHO cells activates multiple G proteins. This was achieved by photoaffinity labeling of G(alpha)-subunits with [32P]azidoanilido-GTP ([32P]AA-GTP) upon hSPR stimulation in CHO-hSPR membranes followed by immunoprecipitation of the labeled G(alpha)-subunits with antibodies specific for various G(alpha)-subunits. These experiments reveal that hSPR directly activates G(alpha q/11), G(alpha s) and G(alpha o). While hSPR is known to couple G(alpha q/11), the present study provides the first evidence that hSPR can also activate G(alpha s) and G(alpha o) in a mammalian system.

Amino Acid Sequence↗
PubMed1985

Cultured neurons contain a variety of microtubule-associated proteins.

The non-tubulin proteins associated with microtubules (MAPs) in cultures of pure sympathetic neurons have been identified using a variety of biochemical and immunochemical methods. MAPs of cultured sympathetic neurons include proteins corresponding to brain MAP-1 (consisting of MAP-1a and MAP-1b species), MAP-2, MAP-3, tau, 4 proteins that range in molecular weight from 60,000 to 76,000, and proteins with molecular weights of 210,000, 130,000 and 32,000. Many of the MAPs are phosphorylated in situ. MAP-2 and tau of cultured sympathetic neurons differ from their counterparts of brain in electrophoretic mobility. The observed variety of MAPs in sympathetic neurons together with the differences in MAPs of brain and sympathetic neurons are discussed in terms of microtubule heterogeneity in the nervous system.

Alkaloids↗
PubMed1993

The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system.

The region of the surface of the histidine-containing protein (HPr) which interacts with the A domain of the mannitol-specific Enzyme II (II(Amt1)) has been mapped by titrating the A-domain into a solution of 15N-labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty-five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of II(Amt1) while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of II(Amt1) resulted in chemical shift changes in a sub-set of the above residues; these were located more in the vicinity of the active site phospho-histidine. Phosphorylation of the HPr/II(Amt1) complex resulted in a HSQC spectrum which was indistinguishable from the P-HPr spectrum in the absence of II(Amt1) indicating that, as expected, the complex P-HPr/P-II(Amt1) does not exist even at the high concentrations necessary for NMR.

Bacterial Proteins↗
PubMed2002

Early and late molecular and morphologic changes that occur during the in vitro transformation of Trypanosoma cruzi metacyclic trypomastigotes to amastigotes.

The amastigogenesis primary of T. cruzi occurs naturally when metacyclic trypomastigotes transform into amastigotes within the cells of the mammalian host. The in vitro study of the macromolecular changes that occur over several days during the transformation process should provide significant indications of how the parasite adapts to the mammalian host environment. We show here that metacyclic trypomastigotes pre-incubated at 37 degrees C in a protein-rich medium reach a high degree of transformation to amastigotes when re-incubated in the fresh medium. Giemsa-stained smears show that during the pre-incubation phase, the metacyclic trypomastigotes undergo lengthening at the posterior end and a thinning out of the entire body. SDS-PAGE analysis of polypeptides and glycopeptides or Western blot with stage-specific antisera analyses indicate that the in vitro primary amastigogenesis is associated with abrupt changes in protein, glycoprotein, and stage-specific antigens that occur simultaneously during the first 24 hours of pre-incubation. Since the differentiating system consists of a rich media at 37 degrees C, temperature and medium constitution must trigger a macromolecular differentiation to amastigotes that precedes the morphological transformation by several days. This transformation is associated with the rearrangement of stage-specific antigens and takes place when the culture medium is changed.

Animals↗
PubMed2005

Potential energy functions for atomic-level simulations of water and organic and biomolecular systems.

An overview is provided on the development and status of potential energy functions that are used in atomic-level statistical mechanics and molecular dynamics simulations of water and of organic and biomolecular systems. Some topics that are considered are the form of force fields, their parameterization and performance, simulations of organic liquids, computation of free energies of hydration, universal extension for organic molecules, and choice of atomic charges. The discussion of water models covers some history, performance issues, and special topics such as nuclear quantum effects.

Biophysics↗
PubMed1981

Reconstitution of binding protein dependent active transport of glutamine in spheroplasts of Escherichia coli.

In order to directly prove that the periplasmic glutamine binding protein is an essential component of the osmotic shock sensitive active transport system for glutamine in Escherichia coli, we demonstrated the reconstitution of binding protein dependent glutamine transport in spheroplasts of that organism. It was shown by arsenate inhibition that the reconstituted transport system was energy dependent, and the use of azaserine, an inhibitor of glutamine-utilizing enzymes, indicated that the restoration of transport by binding protein did not require the metabolizing of the transport substrate. Furthermore, the binding protein dependent transport of glutamine was shown to require at least one other macromolecular component, presumably membrane bound, which was absent in a strain containing a deletion of the genes coding for the glutamine transport system but was present in a strain carrying a mutation only in the structural gene for the glutamine binding protein.

Arsenates↗
PubMed2004

Characterization of macromolecular heterogeneity by equilibrium sedimentation techniques.

New graphical procedures have been developed to investigate the heterogeneity of protein preparations using sedimentation equilibrium. The heterogeneous systems that can be studied include self-associating systems contaminated by incompetent monomer, self-associating systems contaminated by non-dissociating oligomer and simple non-interacting monomer-oligmer disperse systems. The new procedures are based on the concentration dependence of the apparent association constants estimated by a non-linear least square fitting program (NONLIN), on the assumption of conservation of mass during sedimentation and on the applications of several standard techniques for statistical inferences of NONLIN estimations. The procedures outlined here can detect various types of heterogeneity, discriminate amongst different types of heterogeneity, estimate the amount of contaminant causing heterogeneity and determine the true equilibrium constant of the self-associating components. The procedures appear to be sensitive, accurate and easily applicable when tested using both protein samples and computer simulated data.

Attention↗
PubMed1997

Native macromolecular heparin proteoglycans exocytosed from stimulated rat serosal mast cells strongly inhibit platelet-collagen interactions.

Mast cells, the major source of tissue heparin, line the vascular system. On stimulation, rat serosal mast cells release soluble heparin proteoglycans (HEP-PGs) of very high molecular weight (7500(K)). We compared the effects of HEP-PGs and standard heparins (average molecular weights, 15,000 and 5,000) on platelet-collagen interactions in vitro. In contrast with the standard heparins, HEP-PGs completely inhibited collagen-induced platelet aggregation and serotonin release in platelet-rich plasma. The inhibition caused by HEP-PGs depended on its macromolecular structure. In flowing blood, HEP-PGs also inhibited platelet deposition on a collagen-coated surface both at low and high shear rates. Although HEP-PGs did not block glycoprotein (GP) Ia/IIa-mediated platelet adhesion, they attenuated subsequent platelet activation and aggregation, as well as fibrinogen binding to platelets after collagen stimulation. HEP-PGs did not bind to platelets but bound tightly to von Willebrand factor (vWf) and enhanced its binding to collagen. Although platelet adhesion at high shear rate and vWf binding to GP Ib after ristocetin stimulation were not markedly affected, HEP-PGs reduced thrombin-induced aggregation and vWf binding to GP IIb/IIIa. These findings imply that activation of vascular mast cells with ensuing secretion of HEP-PGs may locally attenuate the thrombogenicity of matrix collagen by inhibiting its platelet-activating capacity.

Animals↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Studies on the molecular weight and association of enzyme I.

Studies were conducted on the physical properties of Enzyme I, the first protein in the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Since values lower than those previously reported for the monomer molecular weight were obtained, experiments were performed to determine whether Enzyme I had been partially degraded during isolation of homogeneous protein. Crude extracts and partially purified and homogeneous protein preparations exhibited identical behavior in crossed immunoelectrophoresis analyses, indicating that the isolated protein represented native, intact Enzyme I. The monomeric subunit of Enzyme I is globular, with a frictional ratio of about 1. Sedimentation equilibrium experiments provided a monomer molecular weight of 57,700 +/- 3,400, and gel filtration studies under denaturing conditions gave a comparable value of 57,000. The values previously obtained from polyacrylamide gel electrophoresis analyses in the presence of sodium dodecyl sulfate varied with the conditions used, but under one set of conditions agreed with those given above. The sedimentation equilibrium studies were conducted at 8 degrees C, in the absence of substrates and cofactor (phosphoenolpyruvate, pyruvate, Mg2+). Under these conditions Enzyme I self-associates, but the association is weak, favoring primarily monomer. Because of solubility limitations, the sedimentation experiments were performed with Enzyme I at an initial concentration of 0.5 mg/ml, providing a concentration distribution of 0.1 to 2 mg/ml. Computer analysis of the results showed that within this concentration range it was not possible to distinguish between two modes of self-association, monomer-dimer and isodesmic. The physiological significance of the results is discussed.

Biological Transport↗
PubMed2005

Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis.

Yersinia pestis, the causative agent of plague, utilizes a type III secretion system (T3SS) to inject effector proteins directly into the cytosol of mammalian cells where they interfere with signal transduction pathways that regulate actin cytoskeleton dynamics and inflammation, thereby enabling the bacterium to avoid engulfment and destruction by macrophages. Type III secretion normally does not occur in the absence of close contact with eukaryotic cells. Negative regulation is mediated in part by a multiprotein complex that has been proposed to act as a physical impediment to type III secretion by blocking the entrance to the secretion apparatus prior to contact with mammalian cells. This complex is composed of YopN, its heterodimeric secretion chaperone SycN-YscB, and TyeA. Here, we report two crystal structures of YopN in complex with its heterodimeric secretion chaperone SycN-YscB and the co-regulatory protein TyeA, respectively. By merging these two overlapping structures, it was possible to construct a credible theoretical model of the YopN-SycN-YscB-TyeA complex. The modeled assembly features the secretion signaling elements of YopN at one end of an elongated structure and the secretion regulating TyeA binding site at the other. A patch of highly conserved residues on the surface of the C-terminal alpha-helix of TyeA may mediate its interaction with structural components of the secretion apparatus. Conserved arginine residues that reside inside a prominent cavity at the dimer interface of SycN-YscB were mutated in order to investigate whether they play a role in targeting the YopN-chaperone complex to the type III secretion apparatus. One of the mutants exhibited a phenotype that is consistent with this hypothesis.

Amino Acid Sequence↗
PubMed1993

Macromolecular solvation energies derived from small molecule crystal morphology.

The morphology of small molecule crystals provides a model for evaluating surface solvation energies in a system with similar packing density to that observed for amino acid residues in proteins. The solvation energies associated with the transfer of methylene and carboxyl groups between vacuum and aqueous phases are estimated to be approx. $40 and -260 cal/A2, respectively, from an analysis of the morphology of succinic acid crystals. These solvation energies predict values for contact angles in reasonable agreement with measurements determined from macroscopic monolayer surfaces. Transfer free energies between vapor and water phases for a series of carboxylic acids are also predicted reasonably well by these solvation energies, provided the surface exposure of different groups is quantitated with the molecular surface area rather than the more traditional accessible surface area. In general, molecular surfaces and molecular surface areas are seen to have important advantages for characterizing the structure and energetics of macromolecular surfaces. Crystal faces of succinic acid with the lowest surface energies in aqueous solution are characteristically smooth. Increasing surface roughness and apolarity are associated with higher surface energies, which suggests an approach for modifying the surface properties of proteins and other macromolecules.

Carboxylic Acids↗