Microwave-assisted protein preparation and enzymatic digestion in proteomics

The combinations of gel electrophoresis or LC and mass spectrometry are two popular approaches for large scale protein identification. However, the throughput of both approaches is limited by the speed of the protein digestion process. Present research into fast protein enzymatic digestion has been focused mainly on known proteins, and it is unclear whether these results can be extrapolated to complex protein mixtures. In this study microwave technology was used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion, which rapidly produces peptide fragments. The peptide fragments were further analyzed by capillary LC and ESI-ion trap-MS or MALDI-TOF-MS. The technique was optimized using bovine serum albumin and then applied to human urinary proteins and yeast lysate. The method enabled preparation and digestion of protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 or 25 min, respectively. Equivalent (in-solution) or better (in-gel) digestion efficiency was obtained using microwave-assisted protein enzymatic digestion compared with the standard overnight digestion method. This new application of microwave technology to protein mixture preparation and enzymatic digestion will hasten the application of proteomic techniques to biological and clinical research.     

Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.

We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air. Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins. The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2. 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua). The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Microwave-assisted specific chemical digestion for rapid protein identification

We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.     

Protein Preconcentration Using Nanofractures Generated by Nanoparticle-Assisted Electric Breakdown at Junction Gaps

Sample preconcentration is an important step that increases the accuracy of subsequent detection, especially for samples with extremely low concentrations. Due to the overlapping of electrical double layers in the nanofluidic channel, the concentration polarization effect can be generated by applying an electric field. Therefore, a nonlinear electrokinetic flow is induced, which results in the fast accumulation of proteins in front of the induced ionic depletion zone, the so-called exclusion-enrichment effect. Nanofractures were created in this work to preconcentrate proteins via the exclusion-enrichment effect. The protein sample was driven by electroosmotic flow and accumulated at a specific location. The preconcentration chip for proteins was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. Nanofractures were formed by utilizing nanoparticle-assisted electric breakdown. The proposed method for nanofracture formation that utilizes nanoparticle deposition at the junction gap between microchannels greatly decreases the required electric breakdown voltage. The experimental results indicate that a protein sample with an extremely low concentration of 1 nM was concentrated to 1.5×10⁴-fold in 60 min using the proposed chip.     

Vinculin, a cytoskeletal substrate of protein kinase C

Vinculin, a cytoskeletal protein localized at adhesion plaques, is a phosphoprotein containing phosphoserine, phosphothreonine, and phosphotyrosine. Vinculin has been previously shown to be a substrate for pp60src, a phosphotyrosine protein kinase, but the kinase(s) responsible for phosphorylation of the other amino acid residues is unknown. The present report examines the phosphorylation of vinculin by various serine- and threonine-specific protein kinases. Only protein kinase C, the calcium-activated phospholipid-dependent protein kinase, phosphorylates vinculin at a significant rate (24 nmol/min/mg) and displays marked specificity for vinculin. Both calcium and phosphatidylserine were required for vinculin phosphorylation by protein kinase C. In addition, both phorbol 12,13-dibutyrate (10 nM) and phorbol 12-myristate 13-acetate (10 nM) stimulated vinculin phosphorylation by protein kinase C at a limiting calcium concentration (10(-6) M). Tryptic peptide analysis revealed two major sites of phosphorylation. One site contained phosphoserine and the other contained phosphothreonine. When compared with tryptic maps of vinculin phosphorylated by src kinase, no overlapping phosphorylated peptides were found. The present findings coupled with the plasma membrane location of both these proteins suggest that vinculin may be a physiologic substrate for protein kinase C.     

Sample Preconcentration Utilizing Nanofractures Generated by Junction Gap Breakdown Assisted by Self-Assembled Monolayer of Gold Nanoparticles

The preconcentration of proteins with low concentrations can be used to increase the sensitivity and accuracy of detection. A nonlinear electrokinetic flow is induced in a nanofluidic channel due to the overlap of electrical double layers, resulting in the fast accumulation of proteins, referred to as the exclusion-enrichment effect. The proposed chip for protein preconcentration was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. This study extends our previous paper, in which gold nanoparticles were manually deposited onto the surface of a protein preconcentrator. In the present work, nanofractures were formed by utilizing the self-assembly of gold-nanoparticle-assisted electric breakdown. This reliable method for nanofracture formation, involving self-assembled monolayers of nanoparticles at the junction gap between microchannels, also decreases the required electric breakdown voltage. The experimental results reveal that a high concentration factor of 1.5×10⁴ for a protein sample with an extremely low concentration of 1 nM was achieved in 30 min by using the proposed chip, which is faster than our previously proposed chip at the same conditions. Moreover, an immunoassay of bovine serum albumin (BSA) and anti-BSA was carried out to demonstrate the applicability of the proposed chip.     

Detection and characterization of calcineurin-like activity in Brassica juncea and its activation by low temperature.

Here we describe a method for detecting calcineurin-like activity in Brassica juncea seedlings. The activity was standardized with respect to all the assay components. The optimum reaction time for the assay was found to be 10 min at 0.75 microM of R II phosphopeptide, a specific substrate for calcineurin. Stimulation of activity by CaM (0.1 microM) and CaCl2 (1 mM) was observed. The enzyme showed maximum activity in 125 mM Tris, 200 mM NaCl and 20 mM MgCl2 solution. The activity was differentially distributed in root, shoot and hypocotyls. It was maximum in roots (2.8 nM PO4 released/mg protein), followed by hypocotyls (0.95 nM PO4 released/mg protein) and cotyledonary leaves (0.85 nM PO4 released/mg protein), respectively. Low temperature (LT) stress treatment (4 degrees C) of short durations (5 and 15 min) showed a substantial increase in the activity. Maximum increase was observed in cotyledonary leaves (34.8%), followed by roots (25.6%) and hypocotyls (5.25%), respectively after LT treatment of 5 min suggesting its probable involvement in early signaling events. Besides, in vitro phosphorylation studies also showed activation of phosphatase by LT. Hence, the study indicates probable involvement of calcineurin-like activity in early cold stress signaling. Moreover, this optimized activity assay could be adopted to detect calcineurin-like activity in other plants.     

Microcolumn capture and digestion of proteins combined with mass spectrometry for protein identification

A procedure has been developed for protein identification using mass spectrometry (MS) that incorporates sample cleanup, preconcentration, and protein digestion in a single-stage system. The procedure involves the adsorption of a protein, or protein mixture, from solution onto a hydrophobic resin that is contained within a microcolumn. Sample loading is accomplished by flowing the protein solution through the microcolumn, where the protein adsorbs to the hydrophobic surface. The protein is digested while still bound to the hydrophobic surface by flowing a buffered trypsin solution through the column bed. The peptide fragments are subsequently eluted for detection by MALDI or ESI-MS. The procedure is demonstrated using dilute protein samples containing high concentrations of salt, urea, and modest amount of sodium dodecyl sulfate relative to protein. Peptide fragments are also detected by MS from a 500 nM bacteriorhodopsin solution digested in a microcolumn. In this case, a combined cyanogen bromide/trypsin digestion was performed in-column. The procedure is applied to the MALDI-MS/MS identification of proteins present in an individual fraction collected by ion exchange HPLC separation of E. coli total cell extract. An additional application is illustrated in the analysis of a human plasma fraction. A total of 14 proteins, which were present in the sample at sub-micromolar concentrations, were identified from ESI-MS/MS. The microcolumn digestion procedure represents the next step toward a system for fully automated protein analysis through capture and digestion of the adsorbed protein on hydrophobic surfaces.     

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils.

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases.     

Characterization of Ca2+/Calmodulin-Dependent Protein Kinase Associated with Rat Cerebral Synaptic Junction: Substrate Specificity and Effect of Autophosphorylation

The Ca2+/calmodulin (CaM)-dependent protein kinase associated with rat cerebral synaptic junction (SJ) was characterized, using the SJ fraction as the enzyme preparation, to clarify the functional significance of the enzyme in situ. The protein kinase was greatly activated in the presence of micromolar concentrations of both Ca2+ and calmodulin (EC50 for Ca2+, 1.0 microM; that for CaM, 100 nM). The Km for ATP was 150 microM. SJ proteins were phosphorylated without a lag time, and the phosphorylation reached its maximum within 2-10 min at 25 degrees C. The endogenous substrates consisted of four major (160K, 120K, 60K, and 51K Mr) and 10 minor proteins. Compared with the endogenous substrate phosphorylation, the phosphorylation of exogenously added proteins (myosin light chains from chicken muscle, casein, arginine-rich histone, microtubule-associated protein-2, tau-protein, and tubulin) was weak, although they are expected to be good substrates for the soluble form of the Ca2+/CaM-dependent protein kinase. Autophosphorylation of the enzyme in SJ inhibited its activity and did not alter the subcellular distribution of the enzyme.     

Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting.     

The use of self-interaction chromatography in stable formulation and crystallization of proteins

This paper reviews the basic principles of the recently developed self-interaction chromatography (SIC) technique with regard to protein solution stability and protein crystallization. It gives experimental protocols for both normal-scale and micro-scale SIC experiments and reviews recent developments and current applications of this novel technique in the biopharmaceutical area. This paper aims to be a benchmark in the further proliferation of this highly effective and fast technology for the rational design of stable aqueous formulations of therapeutic proteins and the determination of solution conditions favoring protein crystallization.     

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.     

Identifying mRNAs bound by RNA-binding proteins using affinity purification and differential display

Many methods are available and widely used to determine specific proteins that bind to a particular RNA of interest. However, approaches to identify unknown substrate RNAs to which an RNA-binding protein binds and potentially regulates are not as common. In this article we describe a technique termed isolation of specific nucleic acids associated with proteins (SNAAP) that allows the identification of mRNAs associated with a protein. Methods are detailed for expressing and purifying fusion proteins that are used to isolate substrate mRNPs employing differential display technology. Lastly, experiments are described to confirm that the RNAs identified are indeed bonafide substrates for an RNA-binding protein. As the number of known RNA-binding proteins increases, of which many are involved in genetic disorders, it is essential that methodologies exist to identify RNA-protein interactions to better understand the manifestation of disease.     

The potent vasodilating and guanylyl cyclase activating dinitrosyl-iron(II) complex is stored in a protein-bound form in vascular tissue and is released by thiols.

We studied the biological activity, stability and interaction of dinitrosyl-iron(II)-L-cysteine with vascular tissue. Dinitrosyl-iron(II)-L-cysteine was a potent activator of purified soluble guanylyl cyclase (EC50 10 nM with and 100 nM without superoxide dismutase) and relaxed noradrenaline-precontracted segments of endothelium-denuded rabbit femoral artery (EC50 10 nM superoxide dismutase). Pre-incubation (5 min; 310 K) of endothelium-denuded rabbit aortic segments with dinitrosyl-iron(II)-L-cysteine (0.036-3.6 mM) resulted in a concentration-dependent formation of a dinitrosyl-iron(II) complex with protein thiol groups, as detected by ESR spectroscopy. While the complex with proteins was stable for 2 h at 310 K, dinitrosyl-iron(II)-L-cysteine in aqueous solution (36-360 microM) decomposed completely within 15 min, as indicated by disappearance of its isotropic ESR signal at gav = 2.03 (293 K). Aortic segments pre-incubated with dinitrosyl-iron(II)-L-cysteine released a labile vasodilating and guanylyl cyclase activating factor. Perfusion of these segments with N-acetyl-L-cysteine resulted in the generation of a low molecular weight dinitrosyl-iron(II)-dithiolate from the dinitrosyl-iron(II) complex with proteins, as revealed by the shape change of the ESR signal at 293 K. The low molecular weight dinitrosyl-iron(II)-dithiolate accounted for an enhanced guanylyl cyclase activation and vasodilation induced by the aortic effluent. We conclude that nitric oxide (NO) produced by, or acting on vascular cells can be stabilized and stored as a dinitrosyl-iron(II) complex with protein thiols, and can be released from cells in the form of a low molecular weight dinitrosyl-iron(II)-dithiolate by intra- and extracellular thiols.     

Reversed-phase high-performance liquid chromatography separation of dimethylaminoazobenzene sulfonyl- and dimethylaminoazobenzene thiohydantoin-amino acid derivatives for amino acid analysis and microsequencing studies at the picomole level

A simple and fast reversed-phase high-performance liquid chromatographic method has been developed for the complete separation of 35 dimethylaminoazobenzene sulfonyl (DABS)-amino acids and by-products. This method allows simultaneous determination of primary and secondary amino acids which can be present in protein and peptide hydrolysates and also detects the presence of cysteic acid, S-sulfocysteine, hydroxyproline, taurine, norleucine, cystine, and delta-hydroxylysine. The precolumn derivatization of amino acids with dimethylaminoazobenzene sulfonyl chloride (DABS-Cl) is simple and quick (10 min at 70 degrees C) and allows the complete reaction of primary and secondary amino acids. The separation of the compounds under investigation is achieved in 25 min using a reversed-phase 3-microns Supelcosil LC-18 column at room temperature. The versatility of the proposed method is documented by amino acid determination on protein samples obtained using different hydrolysis techniques (HCl, methane-sulfonic acid, and NaOH), with attention given to the detection of tryptophan in protein samples with high sugar concentration. Furthermore, we have reported the experimental conditions necessary to apply this method to the amino acid analysis of very low amount of proteins (1 to 5 micrograms) electroeluted from a stained band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The stability of DABS-derivatives, the short time of analysis, the high reproducibility and sensitivity of the system, and the complete resolution of all compounds of interest make this method suitable for routine analysis. Furthermore, we have also developed a fast reversed-phase high-performance liquid chromatographic method for the complete separation of dimethylaminoazobenzene thiohydantoin (DABTH)-amino acids. The separation of the compounds under investigation is obtained, at room temperature, in less than 18 min using a reversed-phase Supelcosil LC-18 DB column, 3-micron particles, and also allows the complete separation of DABTH-Ile, DABTH-Leu, and DABTH-Norleu. The short time of analysis, together with the high reproducibility of the system and its sensitivity at picomole levels, make this method very suitable for the identification of DABTH-amino acids released during microsequencing studies of proteins and peptides with the dimethylaminoazobenzene isothiocyanate reagent. In addition, we have shown that it is possible to obtain complete separation of DABTH-amino acids also under isocratic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein

Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.