Cross-linking of the proteins in the outer membrane of Escherichia coli.

1. The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A. Both forms of this protein, proteins A1 and A2, produced similar cross-linking products. No cross-linking of protein A to the peptidoglycan was detected. 2. The proteins of the isolated outer membrane varied in their ease of cross-linking. The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions. The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked. No linkage of protein A to protein B was detected. 3. Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. 4. These results suggest that specific protein-protein interactions occur in the outer membrane.     

Alteration of the ATG start codon of the A protein of bacteriophage phi X174 into an ATT codon yields a viable phage indicating that A protein is not essential for phi X174 reproduction

Bacteriophage phi X174 gene A encodes two proteins: the gene A protein and the smaller A protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A protein in the life cycle of phi X174, however, is unknown. Using oligonucleotide-directed mutagenesis a viable phi X174 mutant was constructed in which the ATG start codon of the A protein was changed into an ATT codon. This mutant, phi X-4499T, does not synthesize A protein. The burst size of phi X-4499T amounted to 50% of that of wild type phi X174. This indicates that A protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage phi X174.     

Regulation of rhodopsin dephosphorylation by arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.     

Isolation of three native forms of myeloperoxidase from human polymorphonuclear leukocytes

The bacteriophage phi X174 gene A encodes two proteins: gene A protein and A* protein. Purified A* protein acts as a single-stranded, DNA-specific endonuclease which remains covalently attached to the 5'-end of the cleavage site. Incubation of A* protein with the synthetic heptamer CAACTTG or with oligonucleotides which yield this heptamer after cleavage with the A* protein yields oligonucleotides with the sequences CAACTTGAG, CAACTTGAGG and CAACTTGAGGA. This indicates that A* protein carries an oligonucleotide with the sequence--AG, -AGG or -AGGA. The oligonucleotide can be transferred to the 3'-end of the heptamer CAACTTG. This suggests that A* protein reacts with a specific DNA sequence in the infected cell.     

Fabrication of self-assembled protein A monolayer and its application as an immunosensor

The self-assembled layer of modified protein A was fabricated. In order to modify protein A, the surface group of protein A was substituted with thiol (-SH) functionality by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and dithiothreitol (DTT). The formation of a self-assembled protein A layer on a Au substrate and its increased binding capacity to antibody were confirmed by surface plasmon resonance (SPR) spectroscopy. The surface structure of self-assembled protein A layer, and the binding status of anti-bovine serum albumin (anti-BSA) and BSA were determined by atomic force microscopy (AFM). Treatment on the self-assembled protein A layer with a detergent, such as Tween 20, increased the binding capacity of anti-BSA, because protein A aggregation was reduced significantly by the detergent; this was confirmed by SPR spectroscopy. The self-assembled layer of chemically modified protein A with enhanced binding capacity can be used for immunosensor fabrication.     

Effect of mannitol and glucose on the distribution and trypsin susceptibility of protein A of Staphylococcus aureus

The biological activity and morphological distribution of protein A on the cell surface were studied in a medium containing an excess of either mannitol or glucose, which suppressed protein A production of Staphylococcus aureus, Cowan I strain. Preculture of the organisms in the presence of a sugar suppressed the expression of protein A, resulting in a decrease in the number of cells bound with antiferritin rabbit IgG molecules, which specifically indicate protein A distribution. The distribution pattern of protein A on the cell surface changed with glucose but not with mannitol. The two-layered ferritin distribution on the organism grown in the control medium was altered into a heavily labeled, thick and rough layer with glucose, suggesting the induction of a conformational change in the polypeptide chain forming protein A. This was positively supported by the increase in trypsin susceptibility of protein A.     

Isolated DNA repeat region from fcrA76, the Fc-binding protein gene from an M-type 76 strain of group A streptococci, encodes a protein with Fc-binding activity

The DNA repeat region of fcrA76, the gene encoding a group A streptococcal Fc-binding protein, was subcloned in-frame into an Escherichia coli plasmid expression vector. The expressed protein product displayed the same Fc-binding properties as the full-length Fc-binding protein expressed from fcrA76. The affinity-purified, full-length Fc-binding protein was found to compete with staphylococcal protein A or streptococcal protein G for binding to beads coated with human IgG. These results are consistent with earlier studies suggesting that the binding sites on human IgG for protein A, protein G and the type II Fc-binding protein from group A streptococci are located at the interface of the CH2 and CH3 domains of the Fc region.     

Immunolabeling efficiency of protein A-gold complexes

A systematic study of the adsorption of protein A on colloidal gold particles varying in size from 5-16 nm was performed at different protein concentrations. The number of protein A molecules bound per colloidal particle was evaluated and the Scatchard analysis of the adsorption parameters was applied for each size of the colloid. The binding of protein A to the colloidal gold surface exhibited the same affinity pattern for all of the particle sizes. At low concentrations of stabilizing protein, adsorption took place with high affinity (Kd 1.96-3.3 nM) and the maximum number of protein A molecules attached with this affinity correlated well with the surface of the particle. At higher concentrations of protein A, adsorption exhibited a significantly lower affinity (Kd 530-800 nM), and no saturation was recorded. Competition by albumin did not reveal a preferential removal of the "low-affinity" bound protein A molecules, contradicting the model of successive shells of stabilizing protein around the colloidal particle. The immunolabeling efficiency of conjugates having the same size of gold nucleus but carrying different numbers of protein A molecules was comparatively investigated by quantitative post-embedding immunocytochemistry. Protein A-gold formed with 5-10-nm colloids gave the highest intensity of labeling when carrying the maximum number of protein A molecules that could be adsorbed with high affinity. Overloading as well as underloading these complexes resulted in a significant decrease of their immunoreactivity. The most efficient conjugates were obtained when stabilization was performed with 6 micrograms protein A/ml gold sol of 5 and 10 nm particle diameter, and 15 micrograms protein/ml of 15-nm colloid.     

Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells.

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.     

Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization.

The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.     

Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains.

Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied. The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains. The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA. The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein. The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition. We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro. The interactions between A and B proteins have been followed by a proteolysis assay. The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein. The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein. In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein. However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond.     

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.     

Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.     

A蛋白定向固定抗体用于椭偏光学生物传感器免疫检测

椭偏光学生物传感器是在椭偏光学显微成像技术的基础上发展的一项生物传感技术。它能够直接观测固体表面上的生物分子面密度,毋需任何标记辅助,适合发展成为一种无标记免疫检测技术。研究了在硅片表面上通过A蛋白定向固定抗体分子用于椭偏光学生物传感器免疫检测的可能性。实验结果表明,通过A蛋白固定抗体得到的抗体膜层的均一性和固定量的重复性能够保证椭偏光学生物传感器免疫检测结果的质量。通过A蛋白定向固定的抗体的抗原结合位点趋向一致,显著提高了抗体与抗原结合的能力。此外,通过蛋白A固定的免疫球蛋白G分子能够结合更多的多克隆抗体分子说明通过A蛋白固定的蛋白质分子能够较好地保持其空间构象。     

Role of polymeric forms of the bacteriophage phi X174 coded gene A protein in phi XRFI DNA cleavage.

Gene A of the phi X174 genome codes for two proteins, A and A* (Linney, E.A., and Hayashi, M.N. (1973) Nature New Biol. 245, 6-8) of molecular weights 60,000 and 35,000, respectively. The phi X A* protein is formed from a natural internal initiator site within the A gene cistron while the phi X A protein is the product of the entire A gene. These two proteins have been purified to homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Previous studies have shown that the phi X A protein is an endonuclease which specifically introduces a discontinuity in the A cistron of the viral strand of supertwisted phi XRFI DNA. In addition to this activity, the phi X A protein also causes relaxation of supertwisted phi XRFI DNA and formation of a phi XRFH DNA . phi X A protein complex which has a discontinuity in the A cistron of the viral strand. This isolatable complex supports DNA synthesis when supplemented with extracts of uninfected Escherichia coli which lack phi X A protein and phi XRFI DNA. The phi XRFII DNA . phi X A protein complex can be attacked by exonuclease III but is not susceptible to attack by E. coli DNA polymerase I, indicating that the 5'-end of the complex is blocked. Attempts to seal the RFII structure generated from the phi XRFII DNA . phi X A protein complex with T4 DNA ligase in the presence or absence of DNA polymerase were unsuccessful. The phi X A protein does not act catalytically in the cleavage of phi XRFI DNA. Under conditions leading to the quantitative cleavage of phi XRFI DNA, the molar ratio of phi XRFI DNA to added phi X A protein was approximately 1:10. At this molar ratio, cross-linking experiments with dimethyl suberimidate yielded 10 distinct protein bands which were multiples of the monomeric phi X A protein. In the absence of DNA or in the presence of inactive DNA (phi XRFII DNA) no distinct protein bands above a trimer were detected. We found it possible in vitro to form a phi XRFII DNA . phi X A protein complex with wild-type phi XRFI DNA (phi X A gene+) and with phi XRFI DNA isolated from E. coli (su+) infected with phage phi X H90 (an am mutant in the phi X A gene). Thus, in vitro, in contrast to in vivo studies, phi X A protein is not a cis acting protein. The purified phi X A* protein does not substitute for the phi X A protein in in vitro replication of phi XRFI DNA nor does it interfere with the action of the phi X A protein which binds only to supertwisted phi XRFI DNA. In contrast, the phi X A* protein binds to all duplex DNA preparations tested. This property prevents nucleases of E. coli from hydrolyzing duplex DNAs to small molecular weight products.     

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.     

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.