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PubMed1989

Affinity labeling of Avena phytochrome with ATP analogs.

The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5'-triphosphate, were used to affinity label purified Avena phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys(75),Ala(25)) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome.

Journal Article↗
PubMed1996

Solubilization, partial purification, and affinity labeling of the membrane-bound isoprenylated protein endoprotease.

A previously described [Ma, Y.-T., & Rando, R. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6275-6279] membrane-associated isoprenylated protein endoprotease is important in the processing of isoprenylated proteins terminating with CAAX. The enzyme is of substantial interest because specific inhibitors of it block the processing and functioning of ras in vivo. The enzyme appears to be an integral membrane protein, as it can only be removed from microsomal membranes with detergent. The enzyme is effectively solubilized by the detergent CHAPSO and can be partially purified (approximately 10-fold) by anion ion exchange and size exclusion chromatography. Attempts to further purify the enzyme by other column means, including affinity chromatography, were unsuccessful. The partially purified enzyme is very sensitive to thiol reagents but insensitive to other kinds of protease inhibitors, suggesting that the enzyme is a thiol protease. Potent and specific chloroketone containing affinity labeling agents have been developed. These novel inactivators owe their potency to an S-farnesylcysteine moiety which is recognized by the enzyme. Specific inhibitors of this type should allow for the identification and cloning of this protease, which is important for signal transduction.

Amino Acid Chloromethyl Ketones↗
PubMed1975

Enzymic synthesis of steroid sulphates. XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-mercuri-17beta-oestradiol.

Oistrogen sulphotransferase (3"-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains asingle sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-mercuri-17beta-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17beta-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17beta-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercure-17beta-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. 2t is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.

Animals↗
PubMed1989

Identification of dexamethasone-binding sites on male-rat liver plasma membranes by affinity labelling.

Binding studies with [3H]dexamethasone identified two binding sites on plasma membranes prepared from the male rat liver, a low-capacity site with a KD of 7.0 nM and a higher-capacity site with a KD of 90.1 nM. Both sites exhibited glucocorticoid responsiveness and specificity for glucocorticoids and progestins. Triamcinolone acetonide, which competes well for the binding of dexamethasone to the cytosolic glucocorticoid receptor, did not compete well for the binding of [3H]dexamethasone to the plasma-membrane binding sites. The binding sites were sensitive to protease and neuraminidase treatment, and resistant to extraction with NaCl, but were extracted with the detergent Triton X-100. As these experiments indicated the presence of plasma-membrane protein components which bind glucocorticoids at physiological concentrations, affinity-labelling experiments with dexamethasone mesylate were conducted. Two peptides were specifically labelled, one at approx. Mr 66,000 and one at Mr 45,000. The Mr-66,000 peptide was not sensitive to glucocorticoids, and was extracted by NaCl, and so did not correspond to either of the sites identified in the dexamethasone-binding studies. The Mr-45,000 entity, on the other hand, resembled the dexamethasone-binding sites in its response to glucocorticoid manipulation of the animal and in its resistance to salt extraction. This peptide was not present in rat serum. Thus we have identified a plasma-membrane peptide which binds dexamethasone. Whether this peptide is involved in transport of the glucocorticoid across the plasma membrane remains to be determined.

Animals↗
PubMed1978

A new affinity label for guanosine nucleotide sites in proteins.

A new guanosine analogue has been synthesized, 5'-p-fluorosulfonylbenzoyl guanosine, which has an electrophilic moiety capable of reacting covalently with several classes of amino acid side chains found in proteins. This compound reacts with bovine liver glutamate dehydrogenase to desensitize it irreversibly to inhibition by GTP, without affecting its intrinsic catalytic activity. The specific addition of GTP or GTP and TPNH to the reaction mixture prevents the loss of sensitivity to GTP inhibition. The corporation of approximately 1 mol of 5'-p-sulfonylbenzoyl guanosine/enzyme subunit is associated with the decreased responsiveness of the enzyme to regulation by GTP. It is proposed that 5'-p-fluorosulfonylbenzoyl guanosine may be reacting within the allosteric GTP site of glutamate dehydrogenase and that this compound may have general applicability in the affinity labeling of regulatory and catalytic sites of proteins which normally bind guanosine nucleotides.

Animals↗
PubMed1975

Versatile properties of a nonsaturable, homogeneous transport system in Bacilus subtilis: genetic, kinetic, and affinity labeling studies.

The mutliphasic kinetics that characterize the transport of many amino acids into Bacillus subtilis suggests a priori at least two mechanisms: (i) a tilis suggests a prior at least two independent transport processes, or (ii) a single, homogeneous system that might involve a negative cooperative mechanism. The highly specific transport system for L-tyrosine and L-phenylalanine in B. subtilis was studied as a case in point. The possible presence of a mixed system of independent transport systems was negated by the rentention of multiphasic kinetics of transport in two types of permease mutants. Furthermore, evaluation of kinetic data obtained during transport under various uptake conditions of pH and temperature, or in the presence of metabolic inhibitors, did not reveal the heterogeneity expected of mechanism (i). These data, taken together with characteristics of substrate specificity and affinity labeling, provide substantial support for a negative cooperative mechanism for L-tyrosine and L-phenylalanine transport.

Amino Acids↗
PubMed2002

Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels.

We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN(2) (I) and Cbz-Leu-Pro-Ala-Thr-CH(2)Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtA(DeltaN)). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (k(i)/K(i)) of 5.3x10(4) M(-1).min(-1), approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN(2) (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.

Aminoacyltransferases↗
PubMed1973

Identification of chloramphenicol-binding protein in Escherichia coli ribosomes by affinity labeling.

Monoiodoamphenicol, a synthetic analogue of chloramphenicol, has been shown by competition experiments with chloramphenicol and lincomycin to bind at the same site of 70S ribosomes as chloramphenicol. At - 2 degrees it forms a 1:1 complex with 70S ribosomes having a value of K (7.5 x 10(4) M(-1)) that is one order of magnitude lower than that of chloramphenicol. At 37 degrees , monoiodoamphenicol irreversibly inhibits the protein-synthesizing activity of E. coli ribosomes. It is shown that the analogue reacted preferentially with protein L16 of E. coli 70S ribosomes, and we therefore conclude that protein L16 belongs to the chloramphenicol-binding site of E. coli ribosomes. Since the chemically reactive group of monoiodoamphenicol resembles iodoacetamide, the reaction of E. coli 70S ribosomes with monoiodoamphenicol was compared to that with iodoacetamide. Iodoacetamide did not react with protein L16, but it predominantly reacted with proteins S18 of the 30S subunit. Furthermore, monoiodoamphenicol was reacted with E. coli ribosomal subunits. Isolated 50S subunits bound monoiodoamphenicol by about one order of magnitude less than 70S ribosomes. Again, protein L16 reacted with the affinity label. Monoiodoamphenicol reacted with protein S18 in isolated 30S subunits; it also bound to 70S ribosomes of Bacillus stearothermophilus, however, it did not bind irreversibly to these 70S ribosomes.

Acetamides↗
PubMed1975

Catechol O-methyltransferase. 6. Affinity labeling with N-haloacetyl-3,5-dimethoxy-4-hydroxyphenylalkylamines.

Several N-acyl-3,5-dimethoxy-4-hydroxyphenylalkylamines have been synthesized and evaluated for their ability to inactive catechol 9-methyltransferase (COMT). N-iodoacetyl-3,5-dimethoxy-4-hydroxyphenylethylamine was found to rapidly and irreversibly inactivate this enzyme. The corresponding N-bromoacetyl derivative also produced inactivation of COMT but at a slower rate than the N-iodoacetyl derivative. The N-acetyl and N-fumaryl derivatives were completely inactive. The inactivation of COMT by these reagents appears to proceed by a unimolecular reaction within a dissociable complex rather than by a nonspecific bimolecular reaction. The proximity of the amino acid residue being modified relative to the site which binds the aromatic portion of these inhibitors was determined using N-iodoacetylphenylakylamines of varying chain length. The number of methylene carbons separating the aromatic ring and the iodoacetamide moiety in these inhibitors did not greatly influence the binding to COMT nor did it affect how rapidly the enzyme was inactivated. From these observations it was concluded that the amino acid moiety being modified by this class of affinity labeling reagents must be relatively close to or part of the site which binds the aromatic region of these inhibitors.

Animals↗
PubMed1982

Affinity labeling of calmodulin-binding components in canine cardiac sarcoplasmic reticulum.

Canine cardiac sarcoplasmic reticulum vesicles have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated a 125I-containing major product of Mr = 40,000, and a minor component of Mr = 120,000. This latter component probably represents a 1:1 complex between the 100,000-dalton Ca2+-ATPase protein and 125I-calmodulin. When cross-linked samples, solubilized in sodium dodecyl sulfate, were boiled for 2 min, the radioactivity associated with the 40,000-dalton component decreased while that associated with components of 26,000 and 28,000 daltons increased. When these boiled samples were stored at -70 degrees C for 1 week, the radioactivity associated with the 26,000- and 28,000-dalton components decreased whereas that associated with the 40,000-dalton component was increased. This suggests that the 40,000-dalton component represents a 1:1 cross-link between the 23,000-dalton form of phospholamban and 125I-calmodulin. The 26,000- and 28,000-dalton cross-linked components probably represent 1:1 cross-links between 125I-calmodulin and the 8,000- and 11,000-dalton subunits, respectively, of phospholamban. A 32P-containing, 40,000-dalton component was formed when dithiobis(succinimidyl propionate) was added to sarcoplasmic reticulum vesicles, phosphorylated in the presence of [gamma-32P]ATP and 3 microM calmodulin. This confirms that the 40,000-dalton affinity-labeled component is a 1:1 cross-link between phospholamban and calmodulin. We propose that phospholamban is the endogenous receptor for calmodulin in cardiac sarcoplasmic reticulum membranes. However, it is unlikely that phospholamban is the endogenous calmodulin-dependent protein kinase in this membrane.

Animals↗