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Biomedical subjects

B T Chait

Publications and source records attributed to B T Chait.

At least 91 records · Page 5Linked to original sources
PubMed1996

In vitro biotransformation of dynorphin A (1-17) is similar in human and rhesus monkey blood as studied by matrix-assisted laser desorption/ionization mass spectrometry.

Dynorphin A (1-17) [Dyn A (1-17)] is an endogenous opioid peptide. In vitro biotransformation of Dyn A (1-17) in human and rhesus monkey blood was studied by matrix-assisted laser desorption/ionization mass spectrometry. Biotransformation was observed to produce various opioid and nonopioid dynorphin A peptides. In this study, in vitro Dyn A (1-17) biotransformation at physiological temperature (37 degrees C) was found to be very similar in human and rhesus monkey blood, although Dyn A (1-17) processing occurred at a faster rate in vitro in monkey blood than in human blood. One dominant pathway in this biotransformation was the slow removal of tyrosine at position one from Dyn A (1-17) to yield the dominant product, Dyn A (2-17). Further slow biotransformation of Dyn A (2-17) also occurred. Another major pathway of Dyn A (1-17) biotransformation is cleavage of the peptide linkage between Arg(6) and Arg(7) to produce the opioid peptide, Dyn A (1-6), and the nonopioid peptide, Dyn A (7-17). These two peptides had a short lifetime in blood, undergoing rapid biotransformation. Our results indicate that the rhesus monkey may be a good model for further in vivo pharmacological and neurobiological studies.

Adult↗
PubMed1995

Weight-reducing effects of the plasma protein encoded by the obese gene.

The gene product of the ob locus is important in the regulation of body weight. The ob product was shown to be present as a 16-kilodalton protein in mouse and human plasma but was undetectable in plasma from C57BL/6J ob/ob mice. Plasma levels of this protein were increased in diabetic (db) mice, a mutant thought to be resistant to the effects of ob. Daily intraperitoneal injections of either mouse or human recombinant OB protein reduced the body weight of ob/ob mice by 30 percent after 2 weeks of treatment with no apparent toxicity but had no effect on db/db mice. The protein reduced food intake and increased energy expenditure in ob/ob mice. Injections of wild-type mice twice daily with the mouse protein resulted in a sustained 12 percent weight loss, decreased food intake, and a reduction of body fat from 12.2 to 0.7 percent. These data suggest that the OB protein serves an endocrine function to regulate body fat stores.

Adipose Tissue↗
PubMed1995

Monitoring reactions of nitric oxide with peptides and proteins by electrospray ionization-mass spectrometry.

Recent studies have demonstrated the biological importance of the interaction of nitric oxide (NO) with proteins. Protein-associated targets of NO include heme, Cys, and Tyr. Electrospray ionization-mass spectrometry was used to monitor the results of exposure of model peptides and an enzyme to NO under different conditions and thus addressed aspects of NO-protein interactions. The molecular mass of a decapeptide containing a single Cys residue increased by 29 Da upon treatment with NO under aerobic and acidic conditions, consistent with the substitution of one NO moiety. The mass of reduced somatostatin, a peptide containing two Cys residues, increased by 58 Da, consistent with the substitution of two NO moieties. These substitutions were prevented by pretreatment of the peptides with N-ethylmaleimide. The strength of the nitrosothiol bond was examined by varying the amount of energy applied to the peptide ions and indicated a labile species. Cys residues were very rapidly nitrosated, while other reactions were observed to occur at much slower rates. These include the further oxidation of nitrosothiol to sulfonic acid and nitration of Tyr. Peptides treated with NO at physiological pH were observed to undergo dimerization as well as nitrosation. These studies were extended to the enzyme p21ras, whose activity has been postulated to be modulated by nitrosothiol formation, and revealed the formation of a single nitrosothiol on p21ras upon NO treatment. These data suggest that electrospray ionization-mass spectrometry allows for quantitation and characterization of nitrosothiol bonds in peptides and proteins.

Amino Acid Sequence↗
PubMed1995

Increased stability of nucleic acids containing 7-deaza-guanosine and 7-deaza-adenosine may enable rapid DNA sequencing by matrix-assisted laser desorption mass spectrometry.

The use of matrix-assisted laser desorption mass spectrometry (MALDI-MS) has been suggested as an ultrafast readout of Sanger DNA sequencing ladders in a manner analogous to that used with sequencing gels. Currently, a serious limitation of MALDI-MS for the analysis of DNA results from the tendency for oligonucleotides to undergo facile fragmentation in the gas phase. The present study was undertaken to gain an understanding of the influence of various chemical structural features of purine bases on the stability of oligodeoxynucleotide ions produced by MALDI. The study focused on the stability of model compounds of the type d(TTTTTTTTTTXTTTTTTTTT TTTT), where T designates deoxythymidine and X a purine-containing 2'-deoxynucleotide. A variety of different purine derivatives were chosen as the base in the nucleotide X. The mass spectra of the model compounds containing 7-deaza analogues of guanine and adenine reveal a significantly increased stability compared to the 7-aza analogues under the conditions of MALDI-MS. The previously reported incorporation of the 7-deaza-2'-deoxy-adenosine triphosphate and the 7-deaza-2'-deoxy-guanosine triphosphate into DNA by polymerases suggests their use in a Sanger dideoxy sequencing experiment. The dideoxy termination products with the 7-deaza-purines instead of the 7-aza-purines might be sufficiently stable to allow separation and detection of the sequencing ladder by MALDI-MS. Thus, an ultrafast (seconds) read-out of DNA sequence may become feasible.

Base Sequence↗
PubMed1995

Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673.

Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.

Amino Acid Sequence↗
PubMed1995

Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry.

A simple biochemical method that combines enzymatic proteolysis and matrix-assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA-binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of the transcription factor Max--a member of the basic/helix-loop-helix/zipper family of DNA-binding proteins. In the absence of DNA and at low ionic strengths, Max is rapidly digested by each of six endoproteases selected for the study, results consistent with an open and flexible structure of the protein. At physiological salt levels, the rates of digestion are moderately slowed; this and the patterns of cleavage are consistent with homodimerization of the protein through a predominantly hydrophobic interface. In the presence of Max-specific DNA, the protein becomes dramatically protected against proteolysis, exhibiting up to a 100-fold reduction in cleavage rates. Over a 2-day period, both complete and partial proteolysis of the Max-DNA complex is observed. The partial proteolytic fragmentation patterns reflect a very high degree of protection in the N-terminal and helix-loop-helix regions of the protein, correlating with those expected of a stable dimer bound to DNA at its basic N-terminals. Less protection is seen at the C-terminal where a slow, sequential proteolytic cleavage occurs, correlating to the presence of a leucine zipper. The results also indicate a high affinity of Max for its target DNA that remains high even when the leucine zipper is proteolytically removed. In addition to the study of the helix-loop-helix protein Max, the present method appears well suited for a range of other structural biological applications.

Amino Acid Sequence↗
PubMed1995

Examination of recombinant truncated mature human fibroblast collagenase by mass spectrometry: identification of differences with the published sequence and determination of stable isotope incorporation.

Human fibroblast collagenase belongs to a family of matrix metalloproteinases which have been implicated in a number of connective tissue disorders ranging from rheumatoid arthritis to tumor invasion. To examine the active site of this enzyme by biophysical studies, a 19 kDa recombinant truncated mature collagenase (mCL-t) was prepared. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been utilized for the characterization of mCL-t. The molecular weights measured by these techniques identified the presence of two closely related protein components separated by approximately 100 Da. Edman sequence analysis demonstrated that the two protein components differ from each other by an amino terminal valine, consistent with the mass spectrometric data. In addition, the molecular weight of mCL-t determined by mass spectrometry did not agree with that calculated from the reported sequence. To identify the origin of this discrepancy, the DNA sequence of the mCL-t clone was examined. Several differences were noted between the DNA sequence of mCL-t and the published collagenase gene sequence. When these differences were taken into account, the measured molecular weights were found to be in good agreement with that calculated for the modified sequence. In separate experiments, both ESI and MALDI mass spectrometry have been used to determine molecular weights of mCL-t samples enriched with stable isotopes 15N and (15N + 13C). The measured molecular weights demonstrated a 97% (15N) and 99% (15N + 13C) incorporation of labeled isotopes in the two samples.

Amino Acid Sequence↗
PubMed1995

A posttranslational modification of beta-actin contributes to the slow dissociation of the spectrin-protein 4.1-actin complex of irreversibly sickled cells.

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin.

Actins↗
PubMed1995

A new neurohypophysial peptide, seritocin ([Ser5,Ile8]-oxytocin), identified in a dryness-resistant African toad, Bufo regularis.

From the pituitary neurointermediate lobe of the African toad Bufo regularis, vasotocin, hydrin 2 (vasotocinyl-Gly) and a mesotocin-like peptide have been isolated by HPLC and characterized by mass spectrometry, amino acid sequence and chromatographic coelution with synthetic peptides. The mesotocin-like peptide has been identified as [Ser5,Ile8]-oxytocin in place of mesotocin ([Ile8]-oxytocin) found in all other amphibians investigated to date. The name seritocin is suggested. The molecule is virtually devoid of oxytocic activity on rat uterus in contrast to mesotocin. On the other hand, the molar ratio of hydrin 2 to vasotocin in the pituitary reaches 2, whereas it is about 1 in toads and frogs from temperate regions. B. regularis is an anuran species able to withstand a hot and dry season by burrowing. The possible relationship between occurrence of seritocin and adaptation to arid environment remains to be demonstrated.

Amino Acid Sequence↗
PubMed1994

Phosphorylation negatively regulates the function of coactivator PC4.

Human positive cofactor 4 (PC4) mediates activator-dependent transcription by RNA polymerase II, apparently through interactions with transcriptional activators and the basal transcription machinery. We report here that PC4 function is modulated by in vivo phosphorylation. Protein-protein interaction studies and in vitro transcription assays demonstrate that only the nonphosphorylated form of PC4 is functionally active. Although recombinant PC4 can be phosphorylated by casein kinase II and protein kinase C in vitro, mutational and mass spectrometric analyses suggest that the in vivo hyperphosphorylation of PC4 is mediated mainly by casein kinase II and restricted to an N-terminal serine-rich region. These observations provide one example of a transcriptional cofactor that is negatively regulated by casein kinase II phosphorylation.

Amino Acid Sequence↗
PubMed1994

Effects of anions on the positive ion electrospray ionization mass spectra of peptides and proteins.

Positive ion electrospray ionization mass spectra of polypeptides are usually obtained from solutions that are acidified and therefore contain relatively high concentrations of anions. The present study describes an investigation of the effects of these ubiquitous anions on the positive ion electrospray ionization mass spectra of peptides and proteins. Certain anionic species in the spray solutions were observed to cause a marked decrease in the net average charge of peptide and protein ions in the mass spectra compared to the average charge measured in the absence of these anions. This charge neutralization effect was found to depend solely on the nature of the anionic species and was independent of the source of the anion (acid or salt), with the propensity for neutralization following the order: CCl3COO- > CF3COO- > CH3COO- approximately Cl-. A mechanism for the observed charge reduction effect is proposed that involves two steps. The first step occurs in solution, where an anion pairs with a positively charged basic group on the peptide. The second step occurs during the process of desolvation or in the gas phase, where the ion pair dissociates to yield the neutral acid and the peptide with reduced charge state. The different propensities for charge neutralization of the different anionic species is presumed to reflect the avidity of the anion-peptide interaction. These findings demonstrate that any attempt to correlate the distribution of charge states observed on proteins in the gas phase (by positive ion electrospray ionization mass spectrometry) with the net charge residing on the protein in solution will require that the described anion effect be taken into account. In addition, it appears that some control over the distribution of charge states on peptides and protein ions can be exercised by an appropriate choice of anion in the electrospray solution.

Acetates↗
PubMed1994

The sigma subunit conserved region 3 is part of "5'-face" of active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase holoenzyme in open promoter complexes were cross-linked to the beta and sigma 70 subunits. Using limited proteolysis and chemical degradation, the cross-link site in sigma 70 was mapped to a segment between amino acids Glu508 and Met561 containing the C-terminal part of conserved region 3. This result, when reconciled with genetic data on the interaction of sigma 70 conserved regions 2 and 4 with the -10 and -35 promoter regions, respectively, allows us to model the orientation of the sigma 70 subunit domains within the open promoter complex.

Amino Acid Sequence↗
PubMed1994

Crystallization of proliferating cell nuclear antigen (PCNA) from Saccharomyces cerevisiae.

Proliferating cell nuclear antigen (PCNA) is the component of the chromosomal DNA replication machinery in eukaryotic cells that confers high processivity upon DNA polymerase delta and epsilon. It has been proposed that PCNA functions by forming a trimeric complex with a ring-like structure through which DNA is threaded. PCNA from the yeast Saccharomyces cerevisiae has been crystallized in a cubic space group (P2(1)3, a = 121.1 A). Unexpectedly, a mercury derivative of PCNA yields crystals that diffract significantly better than crystals of the unmodified protein (2.4 A and 3.0 A resolution, respectively). Mass spectrometry reveals that the derivative results from the addition of two mercury atoms to the protein. Although crystals of the mercurated protein show evidence of non-isomorphism, the anomalous diffraction signal is strong and phases may be determined by multi-wavelength anomalous diffraction (MAD phasing).

Crystallization↗
PubMed1994

Structural and functional properties of region II-plus of the malaria circumsporozoite protein.

During feeding, infected mosquitos inject malaria sporozoites into the host circulation. Within minutes, the parasites are found in the liver where they initiate the first stage of malaria infection. All species of malaria sporozoites are uniformly covered by the circumsporozoite protein (CS), which contains a conserved COOH-terminal sequence called region II-plus. We have previously shown that region II-plus is the parasite's hepatocyte-binding ligand and that this ligand binds to heparan sulfate proteoglycans (HSPGs) on the hepatocyte membrane. Using a series of substituted region II-plus peptides, we show here that the downstream basic amino acids as well as the interdispersed hydrophobic residues are required for binding of CS to hepatocyte HSPGs. We also show that this positively charged stretch of amino acids must be aggregated in order to bind to the receptor. On the basis of this information, we have synthesized a multiple antigen peptide that mimics the hepatocyte-binding ligand. This construct inhibits both CS binding to HepG2 cells in vitro as well as CS clearance in mice.

Amino Acid Sequence↗
PubMed1994

Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe.

The catalytic domain of Schizosaccharomyces pombe casein kinase-1 (the product of the cki1 gene) has been overexpressed in Escherichia coli, purified by chromatographic methods, characterized in vitro, and crystallized in the presence and absence of nucleotide substrate. The best crystals belong to the trigonal space group P3(1)21 or its enantiomorph, have unit cell parameters a = b = 79 A, c = 121 A, and diffract x-rays to 2.0-A resolution. Kinetic characterization of the purified catalytic domain and other C-terminal deletion mutants of Cki1 suggests that it is subject to two forms of regulation. One mechanism involves autophosphorylation, and results in a 4-fold decrease in the affinity for protein substrate. In contrast, truncation of intact Cki1 results in a 3-fold activation in its catalytic rate. This activation may arise from the removal of an inhibitory domain present in the intact enzyme.

Amino Acid Sequence↗
PubMed1994

Matrix-assisted laser desorption mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis: determination of phosphorylation in synapsin I.

A technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2-dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys-C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix-assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (+/- 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2-dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin-dependent protein kinases, which appear to control their biological activity.

Amino Acid Sequence↗
PubMed1994

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.

Amino Acid Sequence↗