Assay of proteins in the presence of interfering materials

Bensadoun, André; Weinstein, David

doi:10.1016/s0003-2697(76)80064-4

Cited by 3,163 publications

(1,048 citation statements)

References 12 publications

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“…Purification of hSULT2A1 was carried out as previously described [36], and the resulting enzyme preparation was homogeneous as judged by SDS-PAGE with Coomassie Blue staining. Protein concentrations were obtained using the modified Lowry procedure [37] with bovine serum albumin as standard. During the purification, catalytic activity of hSULT2A1 was determined by a standard paired ion extraction assay [38, 39].…”

Section: Methodsmentioning

confidence: 99%

Binding interactions of hydroxylated polychlorinated biphenyls (OHPCBs) with human hydroxysteroid sulfotransferase hSULT2A1

Ekuase

Lehmler

Robertson

et al. 2014

Chemico-Biological Interactions

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Polychlorinated biphenyls (PCBs) are persistent environmental contaminants, and exposure to PCBs and their hydroxylated metabolites (OHPCBs) has been associated with various adverse health effects. The mammalian cytosolic sulfotransferases (SULTs) catalyze the sulfation of OHPCBs, and the interaction of OHPCBs with both the SULT1 and SULT2 families of these enzymes has received attention both with respect to metabolic disposition of these molecules and the potential mechanisms for their roles in endocrine disruption. We have previously shown that OHPCBs interact with human hydroxysteroid sulfotransferase hSULT2A1, an enzyme that catalyzes the sulfation of dehydroepiandrosterone (DHEA), other alcohol-containing steroids, bile acids, and many xenobiotics. The objective of our current studies is to investigate the mechanism of inhibition of hSULT2A1 by OHPCBs by combining inhibition kinetics with determination of equilibrium binding constants and molecular modeling of potential interactions. Examination of the effects of fifteen OHPCBs on the sulfation of DHEA catalyzed by hSULT2A1 showed predominantly noncompetitive inhibition patterns. This was observed for OHPCBs that were substrates for sulfation reactions catalyzed by the enzyme as well as those that solely inhibited the sulfation of DHEA. Equilibrium binding experiments and molecular modeling studies indicated that the OHPCBs bind at the binding site for DHEA on the enzyme, and that the observed noncompetitive patterns of inhibition are consistent with binding in more than one orientation to more than one enzyme complex. These results have implications for the roles of SULTs in the toxicology of OHPCBs, while also providing molecular probes of the complexity of substrate/inhibitor interactions with hSULT2A1.

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Section: Methodsmentioning

confidence: 99%

Binding interactions of hydroxylated polychlorinated biphenyls (OHPCBs) with human hydroxysteroid sulfotransferase hSULT2A1

Ekuase

Lehmler

Robertson

et al. 2014

Chemico-Biological Interactions

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“…TCA precipitation of proteins and determination of protein concentration were performed according to published procedures (Bradford, 1976;Bensadoun and Weinstein, 1976). SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (1970).…”

Section: Miscellaneousmentioning

confidence: 99%

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

Hawlitschek

Schneider

Schmidt

et al. 1988

Cell

376

185

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SummaryTransport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of aminoterminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. lO,OOO-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about l&fold more abundant in mitochondria than MPP It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase,' different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.

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“…Protein concentration was determined by the method of Lowry et al (1951) as modified by Bensadoun and Weinstein (1976) using bovine serum albumin for standard protein.…”

Section: Immunoblottingmentioning

confidence: 99%

Changes in ubiquitin and ubiquitin-protein conjugates in the CA1 neurons after transient sublethal ischemia

Hayashi

Takada

Matsuda

1991

Molecular and Chemical Neuropathology

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ABSTRACTlibiquitin is involved in the degradation of denatured proteins in the recovery process after various stresses. To clarify the different responses of the ubiquitin system in the hippocampal neurons after ischemia, we chose 7.5 min of sublethal forebrain ischemia in the rat. After 7.5 min of ischemia, ubiquitin-like immunoreactivity (UIR) in most of the hippocampal pyramidal cells, except for the interneurons, diminished after 3 h of reperfusion, but enhanced UIR and subsequent recovery of UIR were observed in the different hippocampal regions after 24 h of reperfusion. The most prolonged recovery of UIR in the hippocampal cells was observed in the CAl neurons after 72 h of reperfusion. Immunoblot analysis of the proteins extracted from CAI region showed that high-mol-wt ubiquitin conjugates (HMWUC) above 40 kDa increased, whereas free ubiquitin and ubiquitinated histone 2A decreased slightly after 4 h and 24 h of reperfusion. At 72 h of reperfusion, HMWUC decreased to the original level and free ubiquitin slightly increased beyond the control level. These results suggested that (1) diminished UIR does not always mean depletion of entire ubiquitin-protein conjugates; (2) even after sublethal ischemia, damaged proteins in the CAI neurons may increase, and it may take a long time for elimination of these proteins.

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Assay of proteins in the presence of interfering materials

Cited by 3,163 publications

References 12 publications

Binding interactions of hydroxylated polychlorinated biphenyls (OHPCBs) with human hydroxysteroid sulfotransferase hSULT2A1

Binding interactions of hydroxylated polychlorinated biphenyls (OHPCBs) with human hydroxysteroid sulfotransferase hSULT2A1

Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein

Changes in ubiquitin and ubiquitin-protein conjugates in the CA1 neurons after transient sublethal ischemia

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