N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.

Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of less than or equal to 3 x 10(-9) M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme-T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug enzyme complex: (See article). While the values of dissociation constant K1 = (k-1/k1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decrease in the order of core enzyme greater than holoenzyme greater than the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.

Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The three-dimensional structure of the ␣-isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 Å resolution by x-ray diffraction techniques. A single ␤-sheet and several long ␣-helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.

Rat phosphatidylinositol transfer protein (PITP) is a 32-kDa protein of 271 amino acids that transfers phosphatidylinositol and phosphatidylcholine between membranes. The alpha isoform of rat PITP was expressed in Escherichia coli and purified in high yields. The purified protein contained 1 mol of phosphatidylglycerol and had a transfer activity for phosphatidylinositol and phosphatidylcholine equal to or greater than that of PITP purified from mammalian brain. Limited protease digestion was used to further define structure, activity, and function relationships in PITP. PITP alone is relatively resistant to digestion by chymotrypsin, trypsin, and Staphylococcus V8 protease but is readily cleaved by subtilisin. Phospholipid vesicles containing phosphatidic acid enhance susceptibility to digestion by all four proteases. In the presence of vesicles, PITP, which migrates as a 36-kDa protein in SDS-polyacrylamide gel electrophoresis, is cleaved rapidly by trypsin to a form that appears to be 2-3 kDa smaller than the native form. The tryptic fragment retains partial phospholipid transfer activity and shows an enhanced affinity for phospholipid vesicles containing phosphatidic acid. Analysis of the tryptic digestion products by immunoblotting, N-terminal sequencing, and electrospray mass spectrometry showed that trypsin cleaves the C terminus of PITP at Arg253 and Arg259. Thus, removal of the C terminus enhances the affinity of PITP for vesicles and results in a dimunition of transfer activity. Overall, the data show that PITP undergoes conformation changes and that the C terminus becomes more accessible to trypsin when bound to vesicles. Hence, the C terminus is not an essential component of the membrane binding site and may be located distal to it.

No abstract

The isolated sigma subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide. The labeled sigma subunit (PM-sigma) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme. When a stoichiometric amount of core enzyme was added to a solution of PM-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma. The kinetics of the interaction between the sigma subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching. A biphasic change of fluorescence intensity with respect to time was observed when PM-sigma was rapidly mixed with an excess of core enzyme. The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of sigma to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed. From the best-fit kinetic parameters, an overall binding constant of less than or equal to 3X10(-10)M was estimated for the PM-sigma core complex, in agreement with that obtained by the fluorimetric titration. In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 degrees C. The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes. The activation energy for the "overall" conformational change was estimated to be 6.7 kcal/mol. The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of sigma subunit with core enzyme presented here further supports the proposition that the sigma subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.

An antiserum against tubulin, NS20, has been previously shown to inhibit anterograde and retrograde axonal transport by 50% in vivo and in vitro. We report here that Protein A purified NS20 antibodies also attenuate sperm motility by 50% in demembranated sea urchin sperm. This inhibition is absorbed out by preincubating the NS20 antibodies with a biochemically purified porcine microtubule preparation, with recombinant Trypanosoma beta- (but not alpha-) tubulin and most specifically, with a 37 amino acid (a.a.) synthetic peptide corresponding to a domain near (but not including) the porcine beta-tubulin C terminus. Furthermore, addition of this beta-tubulin peptide alone is sufficient to attenuate motility by 50% in demembranated sperm, indicating that this critical 37a.a. NS20 antigen is a motor binding domain. Together, the results suggest that at least two phenotypically distinct forms of microtubule-based motility, axonal transport and flagellar beating, are homologous at the fundamental level of the microtubule domains (the beta-tubulin peptide and we suggest a distinct but similarly located alpha-tubulin domain) mediating the attachment of tubulin-associated motors.

The effects of several organic and inorganic nitrogen compounds on nitrogenase mRNA and enzyme activity levels were examined in anaerobic cultures ofAnabaena variabiis 29413. Even low concentrations of exogenous ammonia (20 ,uM) Nitrogen-fixing microorganisms synthesize nitrogenase and the other proteins required for nitrogen fixation only under conditions in which nitrogen fixation is required to support growth. In the presence of ammonia and certain other fixed nitrogen compounds, the genetic capacity to fix nitrogen is not expressed. The effects of a variety of inorganic and organic nitrogen compounds such as amino acids and amino acid analogs on nitrogenase synthesis and activity have been studied in several nitrogen-fixing bacteria and cyanobacteria. Overall, the results of many studies show that other utilizable nitrogen sources are usually assimilated in preference to N2, and in the presence of many such compounds nitrogenase activity is absent or greatly reduced (1,10,27). In cyanobacteria, formation of heterocysts is also repressed by compounds which repress nitrogenase synthesis (10, 27). In most of the studies referred to above, investigators have measured nitrogenase activity (using the acetylene reduction technique), so that effects on nif gene regulation were not measured directly. In Klebsiella pneumoniae (3, 5) and Anabaena sp. strain 7120 (11), DNA-RNA hybridization techniques have been used to show that ammonia represses nif gene mRNA levels.As described in the preceding paper (12), we have defined conditions for studying derepression of nitrogenase genes of Anabaena variabilis under anaerobic conditions. This permits rapid experiments to be performed without the complication of heterocyst development. As we describe in this paper, we have used this anaerobic system to study the effects of various nitrogen compounds on nitrogenase gene expression at both transcription and enzyme activity levels. Our results support the conclusion that nitrogenous compounds influence nitrogenase levels primarily at the transcription level, probably by repressing transcription initiation, although some compounds may also have indirect effects on enzyme activity. Also, in agreement with most other studies that have not measured mRNA levels, we find that ammonia itself does not appear to be the immediate effector of nif gene expression. MATERIALS AND METHODSMedia and growth conditions. A. variabilis ATCC 29413 was used for all studies. Cultures were grown as described previously (14).Derepression experiments. Preparation of anaerobic cultures and incubation conditions for derepression experiments were as described previously (12). For experiments with NH4C1, KNO3, glutamine, and glutamate, a series of serum vial cultures was prepared containing cells in media (AA/8 with 0.5% fructose and 10 ,uM dichlorophenyldimethylurea) plus desired concentrations of the test compound; each experiment included a control vial containing no addition. The compound being tested was present throughout the experiment.Because carbamyl...