Identification of a bacterial inhibitor of protein kinases. Mechanism and role in host cell invasion.

We show that Escherichia coli produce a factor that inhibits the activity of tyrosine and serine/threonine protein kinases. The factor is a protein found in the periplasmic compartment and is also secreted into the culture medium. Using a particle concentration fluorescence immunoassay specific for tyrosine kinase activity and inhibition of the tyrosine kinase p56(lck), we purified this factor to apparent homogeneity. Analysis of trypsin-digested fragments by mass spectrometry identified the inhibitor as the bacterial periplasmic protein UDP-sugar hydrolase, an enzyme with potent and nonspecific 5'-nucleotidase activity. Overexpression of the enzyme in bacteria leads to coordinate increases in both 5'-nucleotidase and p56(lck) inhibitory activity, confirming the identity of the inhibitor. The kinase inhibitory activity appears to be due to the formation of adenosine, which we show is inhibitory for p56(lck), cAMP-dependent protein kinase, and casein kinase. Overexpression of UDP-sugar hydrolase leads to an increase in the recovery of enteropathogenic E. coli following infection of HeLa cell monolayers and corresponding alterations in tyrosine-phosphorylated host proteins. These results suggest that UDP-sugar hydrolase may be an important factor affecting host cell function following intracellular bacterial infection.

Synthesis of the protein-sequencing reagent 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate and characterization of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins.

We report the synthesis and structural characterization of the novel Edman-type protein-sequencing reagent 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate. A panel of thiohydantoins prepared from this reagent were found stable during liquid chromatography-electrospray mass spectrometry and were detectable at the low femtomole sensitivity level. Furthermore, the signal detected for these compounds in the mass spectrometer was linear from the low femtomole to the low picomole range. The derivatives showed uv absorbance spectra comparable to their phenylthiohydantoin counterparts. The extinction coefficient for the 4-(3-pyridinylmethylaminocarboxypropyl) phenyl thiohydantoin tyrosine was determined by adsorptive sequence analysis of a synthetic pentapeptide featuring an N-terminal 125I-labeled tyrosine. The sequence data suggest that the reagent will be useful for extended sequence analysis of proteins and peptides using commercially available gas-liquid-phase sequencers.

Liquid chromatography-electrospray ionization mass spectrometry of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins.

We report the separation of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by microbore reverse-phase high-performance liquid chromatography and their detection by on-line electrospray ionization mass spectrometry. These compounds are the products of the chemical stepwise degradation of polypeptides using 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate. We describe chromatographic conditions for on-column concentration of the analytes and for baseline separation of the isobaric amino acid derivatives of leucine and isoleucine. A commercially available protein sequencer was readily interfaced with the described analytical system and used for adsorptive sequence analysis of a panel of synthetic peptides containing collectively all 20 naturally occurring amino acids. On-line mass analysis of derivatives generated by automated sequencing confirmed that the derivatives were of the predicted mass and were detectable at comparable signal strength and sensitivity. Finally, we demonstrate that the additional selectivity in data interpretation provided by mass analysis dramatically improves the signal-to-noise ratio and therefore enhances the ability to conclusively interpret protein and peptide sequence data.

Alpha A- and alpha B-crystallin in the retina. Association with the post-Golgi compartment of frog retinal photoreceptors.

alpha A- and alpha B-Crystallins are significant contributors to maintaining the transparency of the vertebrate lens. We have found that both alpha A- and alpha B-crystallins are also present, at approximately equimolar concentrations, in frog retinal cells. They were identified by sequencing portions of each polypeptide, by immunochemical cross-reactivity with antibodies to bovine alpha-crystallins, and by their relative mobility in two-dimensional gel electrophoresis. Retinal alpha-crystallins form macromolecular multimeric complexes similar to those found in the lens, and they are abundant both in soluble and membrane-associated forms. A surprising finding is that alpha-crystallins bind specifically to the photoreceptor post-Golgi membranes that mediate transport of newly synthesized rhodopsin. Upon treatment of post-Golgi membranes with urea or Triton X-114, a portion of the bound alpha B-crystallin remains tightly associated, indicating that the alpha B-form may mediate membrane binding of an alpha-crystallin multimeric complex. Both subunits are synthesized in vitro by isolated frog retinas, but alpha B-crystallin appears to have a higher renewal rate. Newly synthesized alpha-crystallins become associated with the post-Golgi membranes concurrently with newly synthesized rhodopsin. Association of alpha-crystallins with newly synthesized rhodopsin suggests that they may participate in photoreceptor outer segment membrane renewal. Our findings implicate an important function for both alpha A- and alpha B-crystallins in the same, extralenticular, tissue.

Purification and identification of tyrosine-phosphorylated proteins from B lymphocytes stimulated through the antigen receptor.

The activation of protein tyrosine kinase (PTKs) and subsequent tyrosine phosphorylation of cellular proteins is a critical initial signal in the response of eukaryotic cells to mitogens, differentiative signals, and other stimuli. A number of PTK substrates have been identified and many of these are components of signal transduction pathways that regulate cell function. However, the majority of proteins that are tyrosine-phosphorylated in response to receptor signaling remain unidentified. As some of these unidentified PTK substrates may also be signal-transducing proteins, their identification and functional characterization is an important objective towards understanding receptor signaling. We describe the development of a comprehensive and general process for the isolation and structural characterization of tyrosine-phosphorylated proteins. The method involves enrichment by anti-phosphotyrosine affinity chromatography, electrophoretic concentration and separation, and proteolytic fragmentation of individual purified phosphoproteins. Resulting peptide fragments are separated by microbore reverse-phase high performance liquid chromatography (RP-HPLC) and a portion of the eluted peptides are subjected to electrospray-mass spectrometry (ES/MS) for accurate determination of peptide masses. Proteolytic fragmentation of a protein produces a characteristic set of peptide masses that can be used to rapidly identify the protein by searching databases containing the peptide mass "fingerprints" for all known proteins. The identity of the protein established by this method can be confirmed by sequence analysis of selected peptides. We have applied this procedure to the analysis of PTK substrates from B lymphocytes that have been stimulated through the B cell antigen receptor (BCR). Signaling by this receptor is involved in the generation of antibodies against foreign molecules (antigens). The BCR activates multiple PTKs which phosphorylate at least 30 different proteins. We have identified several of these tyrosine-phosphorylated proteins, including Syk, a PTK that is known to be tyrosine-phosphorylated in activated B cells. Thus, the procedure described here can be used to identify regulatory proteins of low abundance. The process consists of a logical succession of compatible steps that avoids pitfalls inherent to prior attempts to characterize low abundance phosphoproteins and should find wide use for the identification of tyrosine-phosphorylated proteins in other cell types.

Structural and functional relationships in two families of beta-1,4-glycanases.

CenA and Cex are beta-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C. fimi-type cellulose-binding domains. CenA and Cex belong to families B and F, respectively, in the classification of beta-1,4-glucanases and beta-1,4-xylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 beta-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p-nitrophenyl-beta-D-xylobioside is 14,385 min-1.mM-1 at 25 degrees C; the corresponding kcat/Km for p-nitrophenyl-beta-D-cellobioside hydrolysis is 296 min-1.mM-1.

Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid-phase sequence analysis.

A method for the determination of the sites of tyrosine phosphorylation in proteins and peptides at the low picomole level for "cold" phosphopeptides and at the subpicomole level for 32P-labeled phosphopeptides is presented. The procedure is based on solid-phase sequence analysis of phosphopeptides immobilized on carrier discs and the "on-line" detection by reverse-phase high-performance liquid chromatography of the phenylthiohydantoin derivative of phosphotyrosine. The procedure is sensitive and automated and allows the identification of phosphotyrosine derivatives in the same operation as the detection of the derivatives of the other common amino acids. Essentially quantitative extraction of the phosphotyrosine derivatives from the sequencer makes this method ideally suited for the quantitative assessment of protein-tyrosine kinase and protein phosphatase activities and for the determination of their respective recognition sequences.

Identification of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein kinase p44mpk.

Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein-serine/threonine kinase (p44mpk) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44mpk from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid-phase sequencing by Edman degradation identified the major site as Thr-97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91-104 in bovine brain myelin basic protein. Thr-94 was also phosphorylated by p44mpk to a very minor extent.

Analysis of dilute peptide samples by capillary zone electrophoresis.

We report a method for the analysis of dilute peptide solutions by capillary zone electrophoresis. The procedure is based on an electrophoretic concentration step of the applied peptide solution in the capillary (stacking) prior to separation, thus allowing the application of increased sample volumes without a breakdown in resolution. Given a constant configuration of the hardware, the method permits the analysis of peptide solutions of an at least 5 times lower concentration than previously possible. The method was applied to the direct analysis of peptide samples separated by narrow-bore reversed-phase high-performance liquid chromatography for high-sensitivity peptide-sequence analysis.

Tunable-diode-laser measurements of gain in optically pumped NH(3).

A tunable-diode laser is used for the first reported time to make gain measurements in a cw optically pumped 12-microm NH(3) amplifier. The tunability of the diode laser enables us to demonstrate that no inversion exists in the 12-microm laser and that Raman processes are responsible for the gain. High-sensitivity techniques allow gain coefficients as low as 0.001%/cm to be detected, and the experimental measurements are found to be in good agreement with theory.

Raman gain in 12-microm NH(3)lasers.

We report the first known observation of lasing in NH (3) on the aP(6, 2) transition at 12.26 microm. Emission is obtained by pumping the aR(4, 2) line with the P(34) 9-microm CO(2) laser line. The frequency offset between pump and absorption line center is 5.16 GHz. This pump-emission pair was chosen to minimize the effects of ac Stark shifts and population transfer and allows us to study the Raman processes occurring in this type of optically pumped laser. We have confirmed that lasing takes place by a Raman process and obtained good agreement between theory and experimental measurements of small-signal gain.