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PubMed2004

Protein microarray system for detecting protein-protein interactions using an anti-His-tag antibody and fluorescence scanning: effects of the heme redox state on protein-protein interactions of heme-regulated phosphodiesterase from Escherichia coli.

A highly sensitive microarray system for detecting protein-protein interactions has been developed. This method was successfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS). To immobilize (His)6-Tag fused Ec DOS, anti-(His)6-Tag monoclonal antibody (anti-(His)6-Tag mAb) was initially immobilized on the solid surface, and (His)6-Tag fused Ec DOS was fixed by antigen-antibody interactions. For this experiment, ProteoChip, generally suitable for antibody immobilization, was used as solid substrate. In this report, we confirm the antibody immobilization ability of ProteoChip and specific binding to the F(c) region of the antibody. Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PAS domain were investigated on the solid surface. Ec DOS immobilized via anti-(His)6-Tag mAb maintained interactions with the PAS fragment, in contrast to directly immobilized Ec DOS in the absence of anti-(His)6-Tag mAb. Heme-redox-sensitive interactions between Ec DOS and the PAS fragment were additionally detected using anti-(His)6-Tag mAb as a mediator. Our results collectively suggest that the immobilization method using anti-Tag antibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures and functions on solid surfaces.

Antibodies↗

PubMed2005

A knowledge-based energy function for protein-ligand, protein-protein, and protein-DNA complexes.

We developed a knowledge-based statistical energy function for protein-ligand, protein-protein, and protein-DNA complexes by using 19 atom types and a distance-scale finite ideal-gas reference (DFIRE) state. The correlation coefficients between experimentally measured protein-ligand binding affinities and those predicted by the DFIRE energy function are around 0.63 for one training set and two testing sets. The energy function also makes highly accurate predictions of binding affinities of protein-protein and protein-DNA complexes. Correlation coefficients between theoretical and experimental results are 0.73 for 82 protein-protein (peptide) complexes and 0.83 for 45 protein-DNA complexes, despite the fact that the structures of protein-protein (peptide) and protein-DNA complexes were not used in training the energy function. The results of the DFIRE energy function on protein-ligand complexes are compared to the published results of 12 other scoring functions generated from either physical-based, knowledge-based, or empirical methods. They include AutoDock, X-Score, DrugScore, four scoring functions in Cerius 2 (LigScore, PLP, PMF, and LUDI), four scoring functions in SYBYL (F-Score, G-Score, D-Score, and ChemScore), and BLEEP. While the DFIRE energy function is only moderately successful in ranking native or near native conformations, it yields the strongest correlation between theoretical and experimental binding affinities of the testing sets and between rmsd values and energy scores of docking decoys in a benchmark of 100 protein-ligand complexes. The parameters and the program of the all-atom DFIRE energy function are freely available for academic users at http://theory.med.buffalo.edu.

Algorithms↗

PubMed2005

Evolution of proteins and gene expression levels are coupled in Drosophila and are independently associated with mRNA abundance, protein length, and number of protein-protein interactions.

Organismic evolution requires that variation at distinct hierarchical levels and attributes be coherently integrated, often in the face of disparate environmental and genetic pressures. A central part of the evolutionary analysis of biological systems remains to decipher the causal connections between organism-wide (or genome-wide) attributes (e.g., mRNA abundance, protein length, codon bias, recombination rate, genomic position, mutation rate, etc) as well as their role-together with mutation, selection, and genetic drift-in shaping patterns of evolutionary variation in any of the attributes themselves. Here we combine genome-wide evolutionary analysis of protein and gene expression data to highlight fundamental relationships among genomic attributes and their associations with the evolution of both protein sequences and gene expression levels. Our results show that protein divergence is positively coupled with both gene expression polymorphism and divergence. We show moreover that although the number of protein-protein interactions in Drosophila is negatively associated with protein divergence as well as gene expression polymorphism and divergence, protein-protein interactions cannot account for the observed coupling between regulatory and structural evolution. Furthermore, we show that proteins with higher rates of amino acid substitutions tend to have larger sizes and tend to be expressed at lower mRNA abundances, whereas genes with higher levels of gene expression divergence and polymorphism tend to have shorter sizes and tend to be expressed at higher mRNA abundances. Finally, we show that protein length is negatively associated with both number of protein-protein interactions and mRNA abundance and that interacting proteins in Drosophila show similar amounts of divergence. We suggest that protein sequences and gene expression are subjected to similar evolutionary dynamics, possibly because of similarity in the fitness effect (i.e., strength of stabilizing selection) of disruptions in a gene's protein sequence or its mRNA expression. We conclude that, as more and better data accumulate, understanding the causal connections among biological traits and how they are integrated over time to constrain or promote structural and regulatory evolution may finally become possible.

Animals↗

PubMed1991

[Determination of kinetic parameters of protein metabolism in growing rats in conjunction with the measurement of energy metabolism. 2. Determination of the energy requirement for protein retention in conjunction with measurement of the protein synthesis rate at different levels of protein supply].

Energy metabolism-by means of indirect calorimetry-and kinetic parameters of the protein metabolism on the basis of the 3-compartment model were measured with 4 groups of 4 or 5 male Wistar rats in the growth range of between 70 and 230 g live weight in a total of 5 alternately successive periods at the feeding levels growth and energy maintenance as well as 4 different levels of protein supply (6, 10, 17 and 26% crude protein in the feed). The partial energy requirement values for protein retention (bp) for every animal and every period are calculated from the data of energy metabolism. On an average of the 3 growth periods they amounted to 1.75 +/- 0.37 kJ/kJ. A statistically significant linear relation with a slope of approximately 1 could be derived regressively between the protein synthesis rate and the protein retention rate, including all 5 test periods. There was no proven relation between the bp values and the corresponding individual values of the ratio of protein synthesis rate-diminished by the regressively derived protein synthesis rate in the N balance-to the protein retention rate. The results do not permit proven statements on the quantitative relations between protein turnover and energy requirement for protein retention, which is first of all due to methodical shortcomings in measuring both protein metabolism and energy metabolism. They indicate, however, that the heat production from protein synthesis has only a relatively low share in the additional energy expenditure for protein retention and does not considerably surpass the necessary minimal cost for the synthesis of the deposited protein in growing rats.

Animal Feed↗

PubMed2004

Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus.

The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.

Binding Sites↗

PubMed2003

Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4.

Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.

Adrenergic alpha-Agonists↗

PubMed2002

Methods for study of protein dynamics and protein-protein interaction in protein-ubiquitination by electron paramagnetic resonance spectroscopy.

Ubiquitination is a post-translation modification whereby the C-terminal end of ubiquitin (Ub) is covalently attached to the amino group of a lysine in a target protein. Additional ubiquitin groups are added using Ub-Ub linkages to form a polyubiquitin chain. A 26S protease complex specifically binds polyubiquitinated proteins and degrades them in an ATP-dependent manner. The target lysine in the substrate protein resides in a domain that is recognized by the ubiquitination machinery in a temporally and spatially controlled manner. The accessibility and the molecular dynamics of the target domain for each protein substrate is expected to be distinctive and this article is intended to facilitate investigations in this uncharted research area of ubiquitination mediated protein turnover by means of site-directed spin labeling. Examples illustrate the methodology of electron paramagnetic resonance data acquisition and interpretation in terms of secondary and tertiary structure resolution of proteins and protein complexes. Analysis of the spin labeled side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin labels allow to model protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. The structural changes accompanying protein function or protein-protein interaction can be monitored in the millisecond time range. These features make site-directed spin labeling an attractive approach for the study of protein--ubiquitin interaction and protein ubiquitination.

Computer Simulation↗

PubMed2006

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins.

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Acute-Phase Proteins↗

PubMed2002

Protein microarrays to detect protein-protein interactions using red and green fluorescent proteins.

Proteomics, the study of protein function on a global scale, will play an important role in furthering our understanding of gene functions, complex biological pathways, and discovery of novel drug targets. A number of techniques have been developed for proteomic studies to identify and analyze proteins, compare protein expression levels, and study protein-protein interactions. Recent developments have applied a DNA array-type approach to immobilize proteins on a surface for high-throughput analysis. Here we report the development and construction of protein chips using derivatized glass and nitrocellulose-coated slides and the employment of recombinant proteins fused with green and red fluorescent proteins for detection. Fluorescent signals were found to be proportional to the amount of arrayed proteins and could be readily detected with a conventional fluorescence slide scanner. This technique allows the investigation of protein-protein interactions without the need for additional labeling steps of probe proteins.

Antibodies↗

PubMed2003

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association.

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/X11beta/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Tissue-specific distribution of a peroxisomal 46-kDa protein related to the 58-kDa protein (sterol carrier protein x; sterol carrier protein 2/3-oxoacyl-CoA thiolase).

The complete sequence of the nonspecific lipid-transfer protein (nsL-TP; sterol carrier protein 2) including the presequence is present at the C-terminus (residues 405-547) of a 58-kDa protein. To be able to study this 58-kDa protein without the interference of nsL-TP, antibodies were raised against predicted epitope regions in the N-terminal part (peptide I, residues 23-43; peptide II, residues 130-149). Using these antibodies, rat tissues were analyzed by immunoblotting. In rat liver, in addition to the 58-kDa protein the antibody against peptide I (alpha-58K23) as well as the antibody against peptide II (alpha-58K130) detected a 46-kDa protein. This suggests that both peptide sequences are present in this 46-kDa protein. Both the 46- and the 58-kDa-proteins were abundantly present in liver and adrenals, but could also be detected in brain, kidney, heart, lung, testes, and ovary. This distribution was observed in tissues from both male and female rats. Immunogold labeling of cryosections of liver showed that alpha-58K23 labels the peroxisomes. From double-labeling experiments using alpha-nsL-TP and alpha-58K23 we conclude that the 46-kDa protein is peroxisomal. We propose that in the peroxisomes the protease that processes pre-nsL-TP also cleaves the 58-kDa protein giving rise to the 46-kDa protein and nsL-TP. In addition to the 58- and 46-kDa proteins, an immunoreactive 44-kDa protein was prominently present in rat heart and at low levels also in small intestine and brain. Immunogold labeling of cryosections of heart and Western blotting of purified mitochondria showed that the 44-kDa protein is localized in the mitochondria. The 44-kDa protein was shown to be identical to mitochondrial sarcomeric creatine kinase, which has a peptide segment of five amino acid residues in common with peptide I.

Acetyl-CoA C-Acetyltransferase↗

PubMed2001

Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6.

PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.

Adaptor Proteins, Signal Transducing↗

PubMed1985

[Protein requirements of young infants. The effect of protein content in food on growth, protein intake and protein utilization].

The protein content of human breastmilk as given by different authors does not agree very well (table I). This may be caused by the stage of lactation and the nutritional state of the mother, which influences the composition of the milk. Likewise there is no agreement in literature concerning the recommended minimum protein level in infant formulae (table II). Generally the protein level in infant formulae is significantly higher than in human breastmilk. In our experiments the growth of new-born infants fed on a formula with a rather low protein level (1,4 g/100 ml) and with an average protein level (1,9 g/100 ml) was compared (table IV). The experimental stage covered the age from 4 weeks up to and including the twelfth week. In the first four weeks of life the infants were breast-fed; after twelve weeks some additional food was given. Both weight and length were recorded of boys as well as girls. No statistically significant differences were observed on the two different formulae. The growthcurves fit very well with the growth of the Dutch infants (graphics). The average weight increase was under all circumstances somewhat higher on the milk with the lower protein content, these differences however were statistically not significant (table V). No significant differences were seen in the food-intake (table VI and VII). The average food efficiency (weight gained per 100 ml of food) was always the highest on the low protein formula. This effect is however not statistically significant. The food efficiency decreased with increasing age. Boys showed a higher food efficiency than girls. The protein intake (grams per kg bodyweight) was of course lower with the low protein formula (table IX). From an average weight increase of about 30 g a day (table V) and the average protein content of the infant, an average protein increment of 3,4 grams a day was calculated. This means that at a body weight of 3500 grams and a milk-intake of 150 ml/kg a day the protein-intake will be respectively 7,4 and 10,0 grams a day with formulae of respectively 1,4 and 1,9 g protein/100 ml. These daily intakes diminished with the daily protein increment of the infant lead to an amount of respectively 7,4 minus 3,4 leaving 4,0 and 10,0 minus 3,4 leaving 6,6 grams of protein that must be metabolized and must be excreted with the urine respectively as 23 mmol and 38 mmol of urea.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Feeding↗

PubMed1999

Purification of a 38-kDa protein from rabbit reticulocyte lysate which promotes protein renaturation by heat shock protein 70 and its identification as delta-aminolevulinic acid dehydratase and as a putative DnaJ protein.

We reported recently that a rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory and p60 and hop by others) functions as a hsp 70 recycling protein and markedly enhances the renaturation of luciferase by hsp 70 (Gross, M., and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70. ADP to hsp 70.ATP, thus enhancing the protein folding activity of hsp 70, is caused by the purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein contaminant. To determine the relationship between RF-hsp 70 and the DnaJ/hsp 40 heat shock protein family, which also enhances protein renaturation by hsp 70, we purified a 38-kDa protein from rabbit reticulocyte lysate based upon its ability to stimulate renaturation of luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa protein has indicated, unexpectedly, that it is the enzyme delta-aminolevulinic acid dehydratase (ALA-D) and that it does not contain detectable sequences corresponding to the DnaJ/hsp 40 protein family. In addition, immunoblot analysis with a polyclonal antibody made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D is about as active in converting delta-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D purified from other sources. Rabbit reticulocyte ALA-D stimulates the renaturation of luciferase by hsp 70 up to 10-fold at concentrations that are the same as or less than that of hsp 70, and it has no renaturation activity in the absence of hsp 70. The renaturation effect of ALA-D is additive with that of RF-hsp 70 at limiting or saturating concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote the dissociation of hsp 70.ADP in the presence of ATP. The renaturation-enhancing effect of ALA-D may be caused by a region near its carboxyl terminus which has sequence homology to the highly conserved domain of the DnaJ protein family, which is similar to the sequence homology between this domain and a carboxyl-terminal region in auxilin, a DnaJ-like protein that requires this region for its hsp 70-dependent function (Ungewickell, E., Ungewickell, H., Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).

Amino Acid Sequence↗

PubMed2004

The human glutathione S-transferase P1 protein is phosphorylated and its metabolic function enhanced by the Ser/Thr protein kinases, cAMP-dependent protein kinase and protein kinase C, in glioblastoma cells.

We report here that the human glutathione S-transferase P1 (GSTP1) protein, involved in phase II metabolism of many carcinogens and anticancer agents and in the regulation of c-Jun NH(2)-terminal kinase-mediated cell signaling, undergoes phosphorylation by the Ser/Thr protein kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), resulting in a significant enhancement of its metabolic activity. GSTP1 phosphorylation by PKA was glutathione (GSH)-dependent, whereas phosphorylation by PKC did not require but was significantly enhanced by GSH. In the presence of GSH, the stoichiometry of phosphorylation was 0.4 +/- 0.03 and 0.53 +/- 0.02 mol incorporated phosphate per mole of dimeric GSTP1 protein. The GSTP1 protein was phosphorylated, in the presence of GSH, by eight different PKC isoforms (alpha, betaIota, betaIotaIota, delta, epsilon, gamma, eta, and zeta), belonging to the three major PKC subclasses, albeit with various efficiencies. The catalytic efficiency, k(cat)/K(m), of the phosphorylated GSTP1 was more than double that of the unphosphorylated protein. In MGR3 human glioblastoma cells, PKA and PKC activation resulted in a significant increase in the level of phosphorylation of the GSTP1 protein and was accompanied by a 2.1- and 2.7-fold increase, respectively, in specific GSTP1 activity in the cells. Peptide phosphorylation analyses and both phosphorylation and enzyme kinetic studies with GSTP1 proteins mutated at candidate amino acid residues established Ser-42 and Ser-184 as putative phospho-acceptor residues for both kinases in the GSTP1 protein. Together, these findings show PKA- and PKC-dependent phosphorylation as a significant post-translational mechanism of regulation of GSTP1 function. The GSH-dependence of the phosphorylation suggests that under high intracellular GSH conditions, such as is present in most drug-resistant tumors, the GSTP1 protein will exist in a hyper-phosphorylated and enzymatically more active state. In normal cells, the functional activation of the GSTP1 protein by PKA- and PKC-dependent phosphorylation could represent a potentially important mechanism of cellular protection, whereas in tumors, increased phase II metabolism of anticancer drugs by the more active phosphorylated GSTP1 protein could contribute to the drug resistance and therapeutic failure frequently associated with increased activities of these Ser/Thr kinases.

Acyltransferases↗

PubMed1993

Method for simultaneous detection of protein kinase A, protein kinase C, protein tyrosine kinase, and calmodulin-dependent protein kinase activities.

We report a simple method that permits simultaneous detection of multiple protein kinase activities using postnuclear supernatant of v-src transformed NIH3T3 cells. A supernatant is incubated with activators of protein kinases and [gamma-32P]ATP, and the phosphorylated proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The method enables detection of activities of at least four protein kinases (protein kinase A, protein kinase C, protein tyrosine kinase(s), and calmodulin-dependent protein kinase III) on a single gel. Experiments using various specific activators and inhibitors of protein kinases indicated that this method can, in crude preparations, reliably detect the protein kinase activities intended for measurement. Protein kinase C activity disappeared when membranes were solubilized, demonstrating the importance of membrane environment for its function. This method should be particularly useful for evaluating and screening protein kinase inhibitors.

3T3 Cells↗

PubMed1990

Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro.

Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with anti-L antibodies, the immunocomplex already showed protein kinase activity. In the presence of P protein, the NP protein was more highly phosphorylated. The results show that Sendai virus L protein possesses a protein kinase activity phosphorylating the other proteins of the viral nucleocapsid in vitro.

Amino Acid Sequence↗

PubMed1995

Protein surface-distribution and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes.

The binding of native cytochrome c to negatively charged lipid dispersions of dioleoyl phosphatidylglycerol has been studied over a wide range of ionic strengths. Not only is the strength of protein binding found to decrease rapidly with increasing ionic strength, but also the binding curves reach an apparent saturation level that decreases rapidly with increasing ionic strength. Analysis of the binding isotherms with a general statistical thermodynamic model that takes into account not only the free energy of the electrostatic double layer, but also the free energy of the surface distribution of the protein, demonstrates that the apparent saturation effects could arise from a competition between the out-of-plane binding reaction and the lateral in-plane interactions between proteins at the surface. It is found that association with nonlocalized sites results in binding isotherms that display the apparent saturation effect to a much more pronounced extent than does the Langmuir adsorption isotherm for binding to localized sites. With the model for nonlocalized sites, the binding isotherms of native cytochrome c can be described adequately by taking into account only the entropy of the surface distribution of the protein, without appreciable enthalpic interactions between the bound proteins. The binding of cytochrome c to dioleoyl phosphatidylglycerol dispersions at a temperature at which the bound protein is denatured on the lipid surface, but is nondenatured when free in solution, has also been studied. The binding curves for the surface-denatured protein differ from those for the native protein in that the apparent saturation at high ionic strength is less pronounced. This indicates the tendency of the denatured protein to aggregate on the lipid surface, and can be described by the binding isotherms for nonlocalized sites only if attractive interactions between the surface-bound proteins are included in addition to the distributional entropic terms. Additionally, it is found that the binding capacity for the native protein is increased at low ionic strength to a value that is greater than that for complete surface coverage, and that corresponds more closely to neutralization of the effective charge (determined from the ionic strength dependence), rather than of the total net charge, on the protein. Electron spin resonance experiments with spin-labeled lipids indicate that this different mode of binding arises from a penetration or disturbance of the bilayer surface by the protein that may alleviate the effects of in-plane interactions under conditions of strong binding.

Chemical Phenomena↗