Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry.

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation.

N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms.

The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.

The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase.

The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P.

Crystallization and preliminary structural results of catalase from human erythrocytes.

Catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, E.C. 1.

Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine.

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide.

The South Amerindian allotype HLA-B*3909 has the largest known similarity in peptide specificity and common natural ligands with HLA-B27.

HLA-B*3909 has only been found among South Amerindians, and presumably arose locally in these populations. It differs from B*3901 by a single Tyr to Ser change at position 99. To analyze the influence of this polymorphism on peptide specificity, pool sequence analysis and sequencing of multiple individual ligands from B*3901 and B*3909 were carried out.

Photoaffinity labeling with the activator IMP and site-directed mutagenesis of histidine 995 of carbamoyl phosphate synthetase from Escherichia coli demonstrate that the binding site for IMP overlaps with that for the inhibitor UMP.

Photoaffinity labeling with IMP was used to attach covalently this activator to its binding site of Escherichia coli carbamoyl phosphate synthetase. We now identify histidine 995 of the large enzyme subunit as the amino acid that is cross-linked with IMP. The identification was carried out by comparative peptide mapping in two chromatographic systems of peptides differentially labeled with [3H]IMP and with the labeled inhibitor [14C]UMP, followed by automated Edman degradation and radiosequence analysis.

Transcription factor AP-2 activity is modulated by protein kinase A-mediated phosphorylation.

We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP-2 [Garcia et al. (1996) J. Neurosci.

High-sensitivity analysis and sequencing of peptides and proteins by quadrupole ion trap mass spectrometry.

This paper describes experience with the commercially available LCQ quadrupole ion trap mass spectrometer applied to the off-line analysis of peptides and proteins. The standard front end of the electrospray probe was replaced with a micromanipulator which, with the aid of a magnifying device, allowed the use of a variety of miniaturized spraying interfaces. The low sample consumption and extended analysis times of these devices were ideally suitable to obtain improved results in terms of sensitivity and mass accuracy.

Carbamate kinase from Enterococcus faecalis and Enterococcus faecium--cloning of the genes, studies on the enzyme expressed in Escherichia coli, and sequence similarity with N-acetyl-L-glutamate kinase.

Carbamate kinase (CK) catalyzes the reversible reaction NH2COO- + ATP <--> NHCOOPO3(2-) + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X-ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs.

An HLA-B27 polymorphism (B*2710) that is critical for T-cell recognition has limited effects on peptide specificity.

The B*2710 subtype differs from the HLA-B27 prototype (B*2705) only by having Glu instead of Val at position 152, in the alpha2 helix of the peptide-binding site. In spite of its structural similarity most alloreactive CTL raised against B*2705 fail to cross-react with B*2710. Indeed, of the residues that are polymorphic among HLA-B27 subtypes, the Val>Glu152 change has the greatest influence on HLA-B27 T-cell antigenicity.

Streptomyces lividans groES, groEL1 and groEL2 genes.

The Streptomyces lividans groES/EL1 operon and groEL2 gene were cloned and their respective DNA sequences determined. The sequenced DNA comprised the genes and their respective regulatory regions in both cases. Transcription of both groES/EL1 and groEL2 seemed to be subjected to temporal control at 30 degrees C.

Anti-brain spectrin immunoreactivity in Alzheimer's disease: degradation of spectrin in an animal model of cholinergic degeneration.

In a previous work, we described the existence of anti-brain spectrin auto antibodies in Alzheimer's disease (AD) patients (J. Neuroimmunol. 68 (1996) 39-44).

HLA-B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket.

B*2701 differs from B*2705-by three amino acid changes: D-->Y74, D-->N77, L-->A81, and from B*2702 only by two: D-->Y74 and T-->I80. Tyr74 is located in the C/F cavity of the peptide-binding site, and is unique to B*2701 among HLA-B27 subtypes. Binding of natural B*2705 and B*2702 ligands to B*2701, and to mutants mimicking subtype changes, was analyzed.

Lack of carboxyl-terminal tyrosine distinguishes the B*2706-bound peptide repertoire from those of B*2704 and other HLA-B27 subtypes associated with ankylosing spondylitis.

B*2704 and B*2706 are two closely related HLA-B27 subtypes, which differ from the common B*2705 by the Asp > Ser77, Val > Glu152, and Ala > Gly211 amino acid changes. In addition, B*2706 differs from B*2704 by the His > Asp114 and Asp > Tyr116 changes. In spite of their similarity B*2704, but not B*2706, was associated to ankylosing spondylitis in a same population.

Purification and characterization of paraoxon hydrolase from rat liver.

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase.

HLA-B27 presents a peptide from a polymorphic region of its own molecule with homology to proteins from arthritogenic bacteria.

A possible mechanism for the pathogenesis of HLA-B27-associated spondyloarthropathies is that peptides from arthritogenic bacteria with homology to endogenous self-peptides presented by HLA-B27, including those derived from HLA-B27 itself, could elicit an autoimmune T-cell response upon infection. We report here that an undecamer corresponding to the polymorphic region of HLA-B27 spanning residues 169-179 is presented in vivo by the B*2701, B*2704 and B*2706 subtypes, but was not detected in the B*2703-bound peptide pool. This peptide binds to B*2705 in vitro with sufficient affinity to allow its natural presentation by this subtype, but it binds with low affinity to B*2703.

Antibodies to human brain spectrin in Alzheimer's disease.

We investigated the existence of antibodies in sera of Alzheimer's disease patients which immunoreact with specific antigens from crude human brain extracts. We found that 49% of patients, per only 5% of control subjects, had increased levels of antibodies to a 240 kDa protein. On the basis of immunological criteria and internal amino acid sequencing, this antigen was identified as brain spectrin, a cytoskeletal protein which appears to be implicated in synaptic plasticity.

Crystallization, characterization, and preliminary crystallographic studies of mitochondrial carbamoyl phosphate synthetase I of Rana catesbeiana.

Carbamoyl phosphate synthetase I (ammonia; E C 6.3.4.

Crystallization, characterization and preliminary crystallographic studies of carbamate kinase of Streptococcus faecium.

Crystals of carbamate kinase (E.C.2.