Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach.

Tryptic digestion of proteins continues to be a workhorse of proteomics. Traditional tryptic digestion requires several hours to generate an adequate protein digest. A number of enhanced accelerated digestion protocols have been developed in recent years.

Crystallization of 5-keto-4-deoxyuronate isomerase from Escherichia coli.

5-Keto-4-deoxyuronate isomerase from Escherichia coli has been crystallized after partial purification. The isomerase was found to be enriched in preparations of an unrelated recombinant protein. Crystals of the isomerase were obtained from two different precipitants despite the fact that the recombinant protein represented roughly 90% of the total protein present.

The rat S-adenosylhomocysteine hydrolase promoter.

The 1.2-kb DNA sequence flanking the transcription start of the AdoHcy hydrolase gene was cloned into the luciferase reporter plasmid pGL3-basic, and promoter activity was measured in transiently transfected CHO cells. Deletion analysis showed that most promoter activity was located within a 153 bp fragment immediately upstream from the predominant transcription start.

The gene and pseudogenes of rat S-adenosyl-L-homocysteine hydrolase.

Two rat liver genomic DNA libraries constructed in lambda DASH and lambda Charon 4A were screened for sequences with similarity to S-adenosyl-L-homocysteine (AdoHcy) hydrolase cDNA. Of 36 clones purified, two contained the AdoHcy hydrolase gene sequence and 34 contained pseudogene sequences. The AdoHcy hydrolase gene, which has been sequenced in its entirety, spans approximately 15 kb and consists of 10 exons.

Expression of rat liver AdoHcy hydrolase in a Rhodobacter capsulatus ahcY mutant restores pigment formation and photosynthetic growth.

An amino acid alignment of fourteen S-adenosylhomocysteine hydrolases shows that sequences from six photosynthetic species and one species possibly derived from algae have an internal 36 to 41 amino acid sequence that is not present in hydrolase sequences from seven non-photosynthetic species. In the photosynthetic eubacterium Rhodobacter capsulatus, the StLB1 strain has a disrupted hydrolase gene, and hydrolase activity is not detectable. Photopigment synthesis and photosynthetic growth are significantly reduced in the StLB1 strain.

The role of cysteine 78 in fluorosulfonylbenzoyladenosine inactivation of rat liver S-adenosylhomocysteine hydrolase.

Inactivation of rat liver S-adenosylhomocysteine hydrolase by the site-directed reagent 5'-p-fluorosulfonylbenzoyladenosine (FSBA) is associated with the formation of a disulfide bond between Cys-78 and Cys-112 (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362; Gomi, T.

Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.

The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.

Site-directed mutagenesis of rat liver S-adenosylhomocysteinase. Effect of conversion of aspartic acid 244 to glutamic acid on coenzyme binding.

Aspartic acid 244 that occurs at the putative NAD(+)-binding site of rat liver S-adenosylhomocysteinase was replaced by glutamic acid by oligonucleotide-directed mutagenesis. The mutant enzyme was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography showed that the purified mutant enzyme was a tetramer as is the wild-type enzyme.

Phosphatidylcholine synthesis in the rat: the substrate for methylation and regulation by choline.

Two lines of evidence led us to reexamine the possibility that methylation of phosphoethanolamine and its partially methylated derivatives, in addition to methylation of the corresponding phosphatidyl derivatives, plays a role in mammalian phosphatidylcholine biosynthesis: (a) Results obtained by Salerno and Beeler with rat [Salerno, D.M. and Beeler, D.

Expression of rat liver S-adenosylhomocysteinase cDNA in Escherichia coli and mutagenesis at the putative NAD binding site.

The cDNA for rat liver S-adenosylhomocysteinase has been cloned, and the nucleic acid sequence has been determined. By comparison of the deduced amino acid sequence for S-adenosylhomocysteinase with that of the dinucleotide binding region for other proteins, the sequence from amino acids 213 to 244 in rat liver S-adenosylhomocysteinase was proposed to be part of the NAD binding site (Ogawa, H., Gomi, T.

Human granulocytes and granulocytes from other species demonstrate differences in chemotactic responsiveness to oxidized N-formyl-methionyl-leucyl-phenylalanine.

1. Oxidation of the methionine of N-formyl-methionyl-leucyl-phenylalanine to the sulfoxide or sulfone derivative results in the loss of the peptide's chemotactic activity for human granulocytes. 2.

Guanine nucleotide-dependent carboxyl methylation of mammalian membrane proteins.

A guanine nucleotide-dependent protein carboxyl methylation is demonstrated in mammalian cell membranes. The methylation of membrane proteins of Mr 20,000-23,000 requires S-adenosylmethionine, GTP or nonhydrolyzable GTP analogs, and a cytoplasmic methyltransferase. The protein methyl groups are stable at neutral pH and under basic conditions hydrolyze to produce methanol.

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum as deduced from the cDNA sequence.

S-Adenosyl-L-homocysteine hydrolase has been cloned from a lambda gt11 cDNA library prepared from Dictyostelium discoideum that had been starved for 3 hours. The sequence of the cloned cDNA was determined and the deduced amino acid sequence was compared to the amino acid sequence of rat AdoHcy hydrolase. When the sequences from the two species were aligned, 74% of the amino acids were in identical positions.

Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line.

The pertussis toxin substrate from RAW264 macrophage cell membranes was characterized by two-dimensional gel electrophoresis and by immunoblots using antibodies directed against different guanine nucleotide binding proteins. RAW264 membranes were found to contain one major pertussis toxin substrate, which was recognized by both antibodies AS/6 and LE/3. The AS/6 antibody was made against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of transducin, and the LE/3 antibody was made against the peptide corresponding to amino acids 160-169 of a guanine nucleotide binding protein (Gi-2-alpha) cloned from a mouse macrophage cell line.

Amino acid sequence of S-adenosyl-L-homocysteine hydrolase from rat liver as derived from the cDNA sequence.

Rat liver cDNA libraries constructed in lambda gt11 were screened for reactivity with polyclonal antibodies to native S-adenosyl-L-homocysteine (AdoHcy) hydrolase (adenosylhomocysteinase; EC 3.3.1.

A human-mouse hybrid cell line that stably expresses chemotaxis to N-formylmethionyl-leucyl-phenylalanine.

Human leukocytes, which contain monocytes and neutrophils that exhibit chemotaxis to fMet-Leu-Phe, were fused with the mouse macrophage RAW264-TG3 cell line, which exhibits chemotaxis to endotoxin-activated mouse serum but not to fMet-Leu-Phe. From such fusions twelve cell lines were isolated, all of which migrated to endotoxin-activated mouse serum. Four of the cell lines also exhibited chemotaxis to fMet-Leu-Phe, and of these cell lines, only one, WBC264-9, retained the capacity to migrate to fMet-Leu-Phe after culture for 20 or more passages.

Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism.

Cholera toxin inhibits chemotaxis of the RAW264 mouse macrophage cell line. The degree of inhibition by cholera toxin increases upon incubation with the cells, suggesting that the entry of the toxin is required for inhibition of chemotaxis. In the absence of guanine nucleotides, cholera toxin catalyzes the [32P]ADP-ribosylation of RAW264 cell membrane proteins of Mr 41,000, Mr 45,000, and a doublet of Mr 48,000-50,000.

Pertussis toxin inhibition of chemotaxis and the ADP-ribosylation of a membrane protein in a human-mouse hybrid cell line.

When WBC264-9C cells are preincubated with pertussis toxin, chemotaxis is inhibited and ADP-ribosylation of a membrane protein with a subunit Mr 41,000 is observed. Both the inhibition of chemotaxis and the ADP-ribosylation by pertussis toxin display a similar time lag, temperature dependence, and pertussis toxin-concentration dependence. Although the inhibition of chemotaxis and the ADP-ribosylation of the membrane protein are qualitatively correlated, nearly complete inhibition of chemotaxis occurs when there is only partial ADP-ribosylation of the membrane protein.

Oxidized N-formylmethionyl-leucyl-phenylalanine: effect on the activation of human monocyte and neutrophil chemotaxis and superoxide production.

F-met-leu-phe, a potent neutrophil and monocyte chemoattractant, can be oxidized to F-met-leu-phe sulfoxide by metabolically activated neutrophils. We investigated the interaction of human neutrophils and monocytes with chemically prepared oxidized derivatives of F-met-leu-phe: F-met-leu-phe sulfoxide and F-met-leu-phe sulfone. We compared the derivatives with nonoxidized parent F-met-leu-phe for specific binding to neutrophils and monocytes, and for effectiveness as chemoattractants and stimuli of superoxide production.

Differentiation of myoblast cell lines and biological methylation: 3-deazaadenosine stimulates formation of multinucleated myofibers.

Treatment of myoblast cell lines with 3-deazaadenosine stimulates differentiation into myofibers. Myoblast clone L5/ 3B5 , which does not form myofibers after 6 days in fusion medium, was stimulated to form myofibers after 5 days of culture in fusion medium containing 50 microM 3-deazaadenosine. Myoblast clone L5/ 3C4 , which normally begins to form myofibers after 4 days in fusion medium, was stimulated by 50 microM 3-deazaadenosine to form myofibers after 3 days in culture and the extent of fusion was also increased.

Chemotaxis and the synthesis of specific proteins are inhibited by 3-deazaadenosine and other adenosine analogs in a mouse macrophage cell line.

It has been shown earlier that 3-deazaadenosine but not 3-deazaaristeromycin inhibits chemotaxis of RAW264 cells (Aksamit, R.R., Falk, W.

Mechanism of the cytostatic activity of 3-deazaaristeromycin, an inhibitor of adenosylhomocysteine hydrolase.

3-Deazaaristeromycin (100 microM) is cytostatic for the RAW264 mouse macrophage cell line. In cells incubated with 3-deazaaristeromycin, Cell replication is effectively stimulated by 0.5 microM homocysteine thiolactone and no further stimulation occurs at concentrations of homocysteine thiolactone greater than 50 microM.