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N F Totty

Publications and source records attributed to N F Totty.

At least 55 records · Page 3Linked to original sources
PubMed1992

Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of grancalcin, a novel 28 kDa EF-hand calcium-binding protein from human neutrophils.

A novel 28 kDa protein, which we have named 'grancalcin', has been identified in human neutrophils. The protein was isolated from the cytosol and found to be a homodimer, with an apparent molecular mass of 55 kDa on gel filtration. Polyclonal antibodies were raised to the native protein. N-Terminal sequence analysis and tryptic-peptide sequence analysis was performed. The protein exhibits sequence similarity to sorcin, a 24 kDa calcium-binding protein over-expressed in certain multi-drug-resistant cell lines. It appears to be a member of the EF-hand family of calcium-binding proteins. The association of a high proportion of this protein with the membranes and granules in the presence of physiological concentrations of calcium may indicate a role in granule-membrane fusion and degranulation.

Amino Acid Sequence↗
PubMed1992

Apolipoprotein AI mutation Arg-60 causes autosomal dominant amyloidosis.

A mutation in the gene for apolipoprotein AI (apoAI) was identified in an English family with autosomal dominant non-neuropathic systemic amyloidosis. The plasma of all affected individuals contained a variant apoAI with one additional charge, as well as normal apoAI. The propositus was heterozygous; the coding region of his apoAI gene contained both the normal sequence and a single-base substitution changing the codon for residue 60 of the mature protein from CTG (leucine) to CGG (arginine). Allele-specific oligonucleotide hybridization showed that the other affected individuals were also heterozygotes and that there was concordance of the mutant allele with the presence of variant plasma apoAI. Amyloid fibrils isolated from the spleen of the propositus consisted of proteins that ran as a doublet with an apparent mass of approximately 10 kDa in SDS/PAGE and a trace band at 28 kDa. Electrospray mass spectrometry of the purified 10-kDa material revealed components with mass corresponding to the N-terminal 88, 92, 93, and 94 residues of apoAI each with substitution of arginine for leucine. These observations were confirmed by direct protein sequencing and laser desorption time-of-flight mass analysis. No material with the normal apoAI sequence was detected. The trace band at 28 kDa yielded the N-terminal sequence of mature apoAI, indicating that intact or minimally degraded apoAI was also present in the fibril preparation. Discovery of this mutation and the detailed characterization of the apoAI fragments that form the amyloid fibrils open additional avenues for investigation of amyloidogenesis.

Amino Acid Sequence↗
PubMed1992

Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit.

Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

Amino Acid Sequence↗
PubMed1992

Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

3T3 Cells↗
PubMed1992

Accelerated high-sensitivity microsequencing of proteins and peptides using a miniature reaction cartridge.

Greatly reduced automated protein sequencing degradation times of less than 25 min with concurrent on-line phenylthiohydantoin (PTH) derivative analysis times of less than 16 min have been achieved by using a miniaturized reaction cartridge with optimized chemical and analytical cycles. Using this method a wide range of standard and novel peptides and proteins have been sequenced with reproducibly high initial and repetitive cycle yields. In these accelerated analyses the recovery of the more labile PTH derivatives was markedly improved by using elevated pressure during cleavage steps and temperature programming throughout the Edman cycle.

Amino Acid Sequence↗
PubMed1991

Three distinct DNA ligases in mammalian cells.

The major DNA ligase of proliferating mammalian cells, DNA ligase I, catalyzes the joining of single strand breaks in double stranded DNA and is active on a synthetic substrate of oligo(dT) hybridized to poly(dA). DNA ligase I does not catalyze the joining of an oligo(dT).poly(rA) substrate. Two additional DNA ligases, II and III, which can act on the latter substrate have been purified from calf thymus. DNA ligase II, which has been described previously, is a 72-kDa protein. DNA ligase III migrates as a 100-kDa protein in denaturing gel electrophoresis. Structural, immunochemical, and catalytic studies on the three DNA ligase activities strongly indicate that they are the products of three different genes.

Adenosine Monophosphate↗
PubMed1991

Identification and functional characterisation of the cellular activating transcription factor 43 (ATF-43) protein.

The promoter motif CGTCA binds multiple cellular factors that mediate a variety of inducible events, including positive responses to raised cellular levels of cAMP and to the Adenovirus E1a protein. To date, at least ten mammalian cDNA clones have been isolated that encode distinct proteins capable of binding to this motif. However, in most cases the precise stimuli that may regulate these different factors have yet to be determined. We have previously shown that the abundant Hela protein ATF-43 forms a complex in vivo with the cyclic AMP response element binding protein (CREB). In this report we definitively show that ATF-43 is the product of the two published cDNA clones, ATF1 and TREB 36. We confirm that ATF1 efficiently heterodimerises with CREB and demonstrate that even though ATF1 and CREB homodimers, as well as the ATF1/CREB heterodimer efficiently bind to the CGTCA motif, the resulting DNA-protein complexes have significantly different stabilities. A region outside the DNA binding domain of ATF1 contributes to the instability of its interaction with DNA. We further show that despite ATF1's homology to CREB, it responds poorly to activation by protein kinase A. In light of our finding that in Hela cells the majority of CREB protein is heterodimerised with ATF1, we speculate on the functional significance of such heterodimers.

Activating Transcription Factors↗
PubMed1991

Identification of a second neutrophil-chemoattractant cytokine generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to melanoma-growth-stimulatory activity.

The intraperitoneal injection of zymosan in the rabbit results in the generation of an inflammatory exudate containing oedema-forming and chemoattractant activities. Previous studies demonstrated the early appearance of the complement fragment C5a, followed by the generation of two mediators related to the cytokine interleukin-8 that were separable by cation-exchange h.p.l.c. N-Terminal amino acid sequencing identified one of these mediators as rabbit interleukin-8. This paper describes the purification of the second cytokine by cation-exchange, gel-filtration and reversed-phase h.p.l.c. The purified material had both oedema-forming and chemoattractant activity when assayed in rabbit skin in vivo. On SDS/PAGE a single 6-8 kDa band was observed and N-terminal amino acid sequencing of the reduced and alkylated protein positively identified 36 amino acids. This sequence revealed the rabbit homologue of melanoma-growth-stimulatory activity. The identification of these two cytokines in vivo will provide an opportunity to investigate the importance of their co-release in the inflammatory process.

Amino Acid Sequence↗
PubMed1991

Location of the active site for enzyme-adenylate formation in DNA ligases.

The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond. Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation. Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme. Comparison of the known primary structures of several other DNA ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology. The ATP-dependent DNA ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids. The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA ligases, whereas the size of the amino-terminal region is highly variable.

Adenosine Monophosphate↗
PubMed1991

Isolation and characterization of a diuretic peptide from Acheta domesticus. Evidence for a family of insect diuretic peptides.

A diuretic peptide (Acheta-DP) has been isolated from extracts of whole heads of the house cricket, Acheta domesticus. The native peptide increases both cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro to an extent comparable with those responses obtained with supra-maximal amounts of crude extracts of corpora cardiaca. The primary structure of Acheta-DP was established as a 46-residue amidated peptide: TGAQSLSIVAPLDVLRQRLMNELNRRRMRELQGSRIQQNRQLLTSI-NH2. Acheta-DP has 41% sequence identity with a diuretic peptide isolated from Manduca sexta, providing direct evidence for the presence of a family of diuretic peptides in insects.

Amino Acid Sequence↗
PubMed1991

Characterization of a diuretic peptide from Locusta migratoria.

A diuretic peptide Locusta-DP, identified by its ability to increase cyclic AMP production in locust Malpighian tubules in vitro, has been isolated and characterized from whole heads of Locusta migratoria. The purified peptide stimulates fluid secretion by Malpighian tubules maximally in vitro. The primary structure of Locusta-DP was established as a 46 residue amidated peptide: MGMGPSLSIVNPMDVLRQRLLLEIARRRLRDAEEQIKANKDFLQQI-NH2. Locusta-DP has 48% sequence identity with Acheta-DP and 49% identity with Manduca-DH, and provides further evidence for the presence of a family of diuretic peptides in insects.

Amino Acid Sequence↗
PubMed1990

A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8.

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.

Amino Acid Sequence↗
PubMed1990

Purification and internal amino acid sequence of the 80 kDa protein kinase C substrate from Swiss 3T3 fibroblasts. Homology with substrates from brain.

The acidic 80 kDa protein kinase C (PKC) substrate was purified from 2.3 x 10(10) Swiss 3T3 fibroblasts. Partial amino acid sequence data were obtained from five peptides generated by S. aureus V8 cleavage of the protein, enabling a total of 91 amino acid residues to be assigned. The sequences of these five peptides were compared to the deduced amino acid sequences of acidic 80-87 kDa PKC substrates from both actively proliferating A431 epidermal carcinoma cells, and fully differentiated neural tissue. Despite their similar physical properties, there was no homology between the peptides derived from the fibroblast 80 kDa protein and the PKC substrate from A431 cells. However, there was 66% homology with the 87 kDa bovine brain protein within the regions covered by the peptides about 30% of the total protein). Furthermore, comparison of the peptides from the fibroblast 80 kDa protein with proteolytic peptides derived from the acidic 80 kDa rat brain protein revealed an overall homology of 89%. These data provide the first direct evidence that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is closely related to the 80-87 kDa PKC substrates detected in fully differentiated neural tissue.

Amino Acid Sequence↗
PubMed1988

Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family.

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.

Amino Acid Sequence↗
PubMed1987

Purification and N-terminal amino acid sequence of the human T lymphocyte CD2 (T11) surface antigen.

CD2 (the sheep erythrocyte receptor) is a surface antigen of human T lymphocytes. A role for CD2 in T cell function has been implicated from the observation that antibodies against CD2 are capable of transmitting both positive and negative signals for cell growth. Biochemically, the molecule has been identified as a broad band on sodium dodecyl sulfate (SDS)-polyacrylamide gels of about 50-58 kDa. This communication demonstrates that CD2 contains N-linked carbohydrate as endo-beta-N-acetylglucosaminidase F digestion reduced its apparent mol. mass to a compact band of 40 kDa. CD2 was purified from the T leukemia cell line J6 by immunoaffinity chromatography and preparative SDS-polyacrylamide gel electrophoresis (PAGE). Four bands of 52, 54, 56 and 58 kDa could be distinguished in the immunoaffinity-purified protein which formed a broad zone on SDS-PAGE extending from about 45 to 58 kDa. Preparative SDS-PAGE yielded a product suitable for determining the N-terminal amino acid sequence. Assignments were made for the first 26 (excluding the 23rd) residues and the sequence identified a novel polypeptide.

Amino Acid Sequence↗
PubMed1987

Purification and identification by amino acid sequence analysis of the subunits of the CD3 antigen of human tonsil T-lymphocytes.

The CD3 antigen was purified from human tonsils by immunoaffinity chromatography and preparative SDS-PAGE; the overall yield was 10%. Amino acid sequence analysis of the separated gamma, delta and epsilon polypeptides revealed that the gamma and epsilon polypeptides were blocked at the N-terminus, whereas the partial N-terminal amino acid sequence for the delta chain was identical to that described by Borst et al. [Nature 312, 455-485 (1984)]. The gamma and epsilon chains were cleaved with formic acid and cyanogen bromide respectively in order to obtain amino acid sequence data. The sequences obtained corresponded exactly to the amino acid sequences deduced from the nucleotide sequences of putative gamma and epsilon cDNA clones.

Amino Acid Sequence↗