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B T Chait

Publications and source records attributed to B T Chait.

At least 109 records · Page 6Linked to original sources
PubMed1994

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.

Amino Acid Sequence↗
PubMed1994

High-accuracy mass measurement as a tool for studying proteins.

Electrospray ionization and matrix-assisted laser desorption/ionization, two new mass spectroscopy methods for the accurate measurement of molecular masses of individual peptide and protein molecules, are finding great utility for the solution of problems in biological research. Thus, mass spectrometry is being used for the rapid identification and detailed characterization of proteins, the determination of modifications in proteins, and the assessment of the integrity and purity of (native, recombinant, or synthetic) protein preparations. Recent data indicate that mass spectrometry can contribute significantly to the study of protein interactions and even to the investigation of aspects of protein folding and conformation.

Animals↗
PubMed1994

Recombinant hemoglobin A produced in transgenic swine: structural equivalence with human hemoglobin A.

Recombinant human hemoglobin A produced by coexpressing human alpha and beta globin genes in swine, and purified from the lysate of transgenic swine has been subjected to detailed protein chemical analysis. These structural studies involving laser desorption mass spectrometry, separation of globin chains by RPHPLC, amino terminal sequence analysis of the isolated globin chains, the tryptic peptide mapping of the purified globin chains and the amino acid composition analysis of the purified tryptic peptides of globin chains have established the primary structural equivalence of the globin chains of the transgenic swine derived hemoglobin A with that of human hemoglobin A. These results demonstrate that the transgenic swine system correctly translates the human alpha and beta globin m-RNA; carries out the correct cotranslational processing of globin chains, and does not introduce any unwanted post translational modifications into the mature chains. Thus, the transgenic swine expression system is an excellent approach for the production of HbA for developing an effective hemoglobin based oxygen carrier.

Amino Acid Sequence↗
PubMed1993

Biochemical and functional properties of recombinant human sickle hemoglobin expressed in yeast.

Previous studies had indicated that recombinant and natural human sickle hemoglobin had similar chemical properties (Martin de Llano, J. J., Schneewind, O., Stetler, G., and Manning, J. M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 918-922). In the present study, additional biochemical and physiological characterization of some primary through quaternary structural features of recombinant sickle hemoglobin are described. The molecular weight of the purified recombinant sickle hemoglobin was identical to natural sickle hemoglobin as determined by mass spectrometry, thus excluding extensive post-translational modification in the yeast system. Carboxypeptidases A and B together catalyzed the release of COOH-terminal amino acids at the same rate for recombinant and natural hemoglobin S, consistent with identity in their primary and secondary structures in this region of the molecule. The tryptic peptide maps of natural and recombinant hemoglobins were practically indistinguishable, indicating the same internal protein sequences for recombinant and natural hemoglobins. As a probe of the secondary structure of recombinant sickle Hb, the reactivity of the SH group of Cys-93(beta) was investigated for the glutathione sickle hemoglobin adduct, which has significant anti-gelling and anti-sickling properties. The position of glutathione at Cys-93(beta) was established by direct mass spectrometric analysis of enzyme digests; reduction of this derivative to the unmodified chains was also observed by mass spectrometry and by isoelectric focusing. The oxygen equilibrium curves of recombinant and natural sickle hemoglobin at high protein concentration were superimposable with identical Hill coefficients of 3.3. The response of recombinant sickle hemoglobin to chloride with respect to a lowered oxygen affinity was identical to that of natural sickle hemoglobin. The gelation properties of recombinant and natural sickle hemoglobins were identical at the high hemoglobin concentrations that occur in the red cell. Therefore, the yeast expression system synthesizes a completely functional recombinant sickle hemoglobin with the same biochemical and physiological properties as natural sickle hemoglobin with respect to features characteristic of its primary through quaternary structures.

Amino Acids↗
PubMed1993

Protein ladder sequencing.

A new approach to protein sequencing is described. It consists of two steps: (i) ladder-generating chemistry, the controlled generation from a polypeptide chain by wet chemistry of a family of sequence-defining peptide fragments, each differing from the next by one amino acid; and (ii) data readout, a one-step readout of the resulting protein sequencing ladder by matrix-assisted laser-desorption mass spectrometry. Each amino acid was identified from the mass difference between successive peaks, and the position in the data set defined the sequence of the original peptide chain. This method was used to directly locate a phosphoserine residue in a phosphopeptide. The protein ladder sequencing method lends itself to very high sample throughput at very low per cycle cost.

Amino Acid Sequence↗
PubMed1993

Isolation and properties of YCK2, a Saccharomyces cerevisiae homolog of casein kinase-1.

A soluble fragment of YCK2, a casein kinase-1 isoform from Saccharomyces cerevisiae, has been purified and characterized in vitro. The procedure enriches enzyme activity to a final specific activity of 4.7 mumol min-1 mg-1 (when assayed with casein as substrate). Structural analysis reveals that the preparation arises from N-terminal modification and C-terminal proteolysis of the initially synthesized 546-residue protein, consisting of residues 2-495 +/- 1. Kinetic analysis demonstrates that YCK2 is similar to casein kinase-1 isolated from other organisms in its inability to use GTP as nucleotide substrate, in its sensitivity to heparin and ribofuranosyl-benzimidazole inhibitors, and in its peptide substrate selectivity. The enzyme is unusual, however, in that it is insensitive to the potent mammalian casein kinase-1 inhibitor N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide.

Amino Acid Sequence↗
PubMed1993

The extracellular hemoglobin of the earthworm, Lumbricus terrestris. Determination of subunit stoichiometry.

The giant extracellular hemoglobin of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, and c (forming a disulfide-linked trimer) and d ("monomer"). Participation of additional "linker" chains L1, L2, and L3 is necessary for the assembly of the approximately 3,900+ kDa two-tiered hexagonal structure. We have determined the proportions of linker chains, trimer, and chain d in the hemoglobin by reverse phase high performance liquid chromatography which resolves all of the components and also permits simultaneous determination of the heme content. The proportions of components were determined by two independent procedures: integration of the absorbance peaks at 220 nm and amino acid analysis of the peak fractions. The results indicate that the weight proportion of linker chains is 0.163 +/- 0.023. This value, together with molecular masses determined both by amino acid sequence analysis and by matrix-assisted laser desorption mass spectrometry, gives a molar ratio of abcd chains to linkers of 8:1, corresponding to the minimal unit (abcd)2.L. This ratio suggests that 24 (abcd)2 units and 24 linker chains form the complete structure with a total calculated mass of polypeptide of 3,975 kDa with hemes on chains a, b, c and d and on one linker. The calculated heme content is 3.1% not including carbohydrate. This accounts for a measured heme content of 3.0% on a polypeptide basis. Additional mass (approximately 133 kDa, 3.4%), attributed to carbohydrate, brings the total mass to 4,108 kDa with a minimum molecular mass/heme of 20,500 Da. The presence of equimolar quantities of three unique linker chains means that the apparent one-twelfth structural units seen by electron microscopy cannot all be identical.

Amino Acid Sequence↗
PubMed1993

Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites.

ChPrP is the chicken homologue of PrPC, the cellular isoform of the mammalian prion protein. We have used sequence-specific antibodies to immunoprecipitate and immunoblot chPrP derived from stably transfected cultures of neuroblastoma cells, as well as from chicken brain and cerebrospinal fluid. We have also used mass spectrometry to characterize fragments of the protein purified from conditioned medium. The majority of chPrP protein present in neuroblastoma cells and on isolated brain membranes can be released by incubation with phosphatidylinositol-specific phospholipase C, indicating that these molecules are attached to the cell surface by a glycosylphosphatidylinositol anchor. Surprisingly, most of the surface-anchored molecules are truncated at their N-terminus distal to the proline/glycine-rich repeats. The corresponding N-terminal fragments are found in medium conditioned by neuroblastoma cells, as well as in cerebrospinal fluid and a postmicrosomal supernatant of brain. One of these fragments extends from Lys25 to Phe116. 35-45-kDa forms of chPrP that can be metabolically labeled with [3H]ethanolamine can also be found in extracellular media. We propose that the chPrP molecule undergoes at least two cleavages as part of its normal metabolism: one within the glycosylphosphatidylinositol anchor and one within or just N-terminal to the central hydrophobic domain. The second cleavage lies within a region of 24 amino acids that is identical in chPrP and mammalian PrP, and represents a major processing event that may have physiological as well as pathological significance.

Animals↗
PubMed1993

Heat-induced conformational changes in proteins studied by electrospray ionization mass spectrometry.

A simple and effective device for investigating heat-induced denaturation of proteins by electrospray ionization mass spectrometry is described. Results are presented for the denaturation as a function of temperature and solution pH of bovine ubiquitin and bovine cytochrome c. These results are in concert with and extend the earlier results of LeBlanc et al. (Org. Mass Spectrom. 1991, 26, 831). The cooperative effects of pH and temperature on the denaturation of ubiquitin and cytochrome c were investigated. Electrospray ionization mass spectrometry is also shown to be a useful probe of the reversibility of heat-induced denaturation of proteins. Finally, it is demonstrated that heat-induced denaturation can be used to improve the mass spectrometric response of proteins that do not normally yield useful spectra when the solubilized protein is electrosprayed at ambient temperatures.

Animals↗
PubMed1992

252Cf plasma desorption mass spectrometry in the synthesis of porphyrin model systems.

252Cf plasma desorption mass spectrometry has been used in the characterization of more than 100 synthetic porphyrins ranging in mass from 614 u for tetraphenylporphyrin to over 2000 u for some porphyrin model systems. In virtually every case, 252Cf plasma desorption mass spectrometry yielded an intense ionized molecule ion [M.+ and/or (M+H)+], irrespective of the groups appended to the porphyrin. The appended groups include carboxylic acids, amides, imides, chloroacetamides, Fmoc-protected amino acids, aromatic amines, nitriles, alkynes, alkenes, esters, active esters, benzyl ethers, acetals, dithioacetals, ketones, imines, phenols, quinone, hydroquinone, ferrocene, cyanine dyes, trimethylsilyl protecting groups, nitro groups, and combinations of these functionalities. Metalloporphyrins and porphyrin-porphyrin dimers are also analyzed with ease. Resolved isotopic peaks were observed for porphyrins with molecular weights below 1000, and unresolved isotopic peaks yielding average masses were observed for porphyrin compounds with higher molecular weights. The limited resolution in the higher molecular weight range does not lessen the utility of the method because the observation of the molecule ions [M.+ and/or (M+H)+] provides unambiguous evidence concerning the success of the synthesis. The 252Cf plasma desorption mass spectra of porphyrins are not complicated by chemical transformations. This method is ideally suited for rapid analysis of synthetic porphyrins and provides a powerful tool for chemists engaged in the synthesis of complex organic molecules.

Californium↗
PubMed1992

Heterodimeric structure of the spider toxin omega-agatoxin IA revealed by precursor analysis and mass spectrometry.

We report the first molecular characterization of a precursor sequence for a small, Ca2+ channel blocking, peptide spider toxin, omega-agatoxin IA. By integrating information generated from a molecular genetic approach using agatoxin cDNAs with data provided from mass spectrometry of the mature toxin, we were able to deduce the likely mechanisms by which the toxin precursor peptide is processed to its mature heterodimeric form. A particularly interesting feature of the prepropeptide is the occurrence of two glutamate-rich sequences interposed between the signal sequences, the major peptide toxin, and the minor toxin peptide. Excision of the more distal glutamate-rich region appears to be signaled by flanking arginine residues but likely occurs only after a disulfide linkage has formed between the major and minor chains of the mature toxin. Our molecular genetic approach toward characterizing this toxin will allow us to quickly generate a series of spider sequences from which mature toxin structures can be deduced and eventually expressed. Additionally, this approach will provide insights into the evolutionary divergence observed among spider peptide toxins.

Agatoxins↗
PubMed1992

Weighing naked proteins: practical, high-accuracy mass measurement of peptides and proteins.

Two new technologies have made the study of proteins by mass spectrometry straight-forward. Proteins with molecular masses of up to more than 100 kilodaltons can be analyzed at picomole sensitivities to give simple mass spectra corresponding to the intact molecule. This development has allowed unprecedented accuracy in the determination of the molecular weights of proteins. A number of "case studies" are used to present the revolutionary impact that these powerful new ways of looking at proteins are having on biological research.

Amino Acid Sequence↗
PubMed1992

Primary structure of Euplotes raikovi pheromones: comparison of five sequences of pheromones from cells with variable mating interactions.

The amino acid sequences of five pheromones, Er-2, Er-3, Er-9, Er-11, and Er-20, secreted by cells of different mating types of the ciliated protozoa Euplotes raikovi, have been determined by automated Edman analyses of the whole proteins and germane fragments. In each case, the molecular mass was determined by plasma desorption or laser desorption mass spectrometry and was in excellent agreement with the calculated values. Where available, the determined sequences were also in accord with the corresponding segments of the precursor molecules predicted from relevant nucleic acid sequences. Of the five, two were found to be identical (Er-2 and Er-9) and one (Er-3) was identical to a pheromone previously sequenced (Er-1), even though mating pair formation was found to take place (although to a limited extent) when cells secreting those pheromones were combined in a mixture. Comparison of the five unique sequences suggested a closer relationship between Er-1 (Er-3) and Er-10 and between Er-11 and Er-20 (44% and 56% identity, respectively) than was generally observed among the other members. This pairing was also supported by hydrophobicity analyses. Interestingly, Er-20 cannot, as a rule, induce cell union in any of the other cell types, including cells secreting Er-11, despite the fact that Er-20 and Er-11 are the most similar of the five unique sequences. Thus sequence identity and secondary structure profiles are not a good indicator of biological relatedness as manifested in heterologous receptor interaction.

Amino Acid Sequence↗
PubMed1992

The disulfide bond pairing of the pheromones Er-1 and Er-2 of the ciliated protozoan Euplotes raikovi.

The disulfide pairings of the two Euplotes raikovi pheromones Er-1 and Er-2 have been determined by chemical and mass spectrometric analyses. Cystine-linked peptides from thermolytic digestions of the native molecules were purified by reverse-phase high performance liquid chromatography and identified in the known sequences to make the assignments. The same pairing, Cys(I)-Cys(IV), Cys(II)-Cys(VI), and Cys(III)-Cys(V), was found in both pheromones, suggesting that this pattern occurs commonly throughout this family of molecules. This arrangement of disulfides indicates that the three-dimensional structure is defined by three loops, which can vary in size and charge distribution from one pheromone to another.

Amino Acid Sequence↗
PubMed1991

The mevalonate pathway in the bloodstream form of Trypanosoma brucei. Identification of dolichols containing 11 and 12 isoprene residues.

The major surface antigen of the bloodstream form of Trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. The biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. Preliminary observations (Menon, A.K., Schwarz, R.T., Mayor, and Cross, G.A.M. (1990) J. Biol. Chem. 265, 9033-9042) suggested that the sugar carriers in T. brucei were short-chain polyisoprenoids containing substantially fewer isoprene residues than polyisoprenols in mammalian cells. In this paper we describe metabolic labeling experiments with [3H]mevalonate, as well as chromatographic and mass spectrometric analyses of products of the mevalonate pathway in T. brucei. We report that cells of the bloodstream form of T. brucei contain a limited spectrum of short chain dolichols and dolichol phosphates (11 and 12 isoprene residues). The total dolichol content was estimated to be 0.28 nmol/10(9) cells; the dolichyl phosphate content was 0.07 nmol/10(9) cells. The same spectrum of dolichol chain lengths was also found in a polar lipid that could be labeled with [3H]mevalonate, [3H]glucosamine, and [3H]mannose, and which was characterized as Man5GlcNAc2-PP-dolichol. The most abundant product of the mevalonate pathway identified in T. brucei was cholesterol (140 nmol/10(9) cells). Ubiquinone (0.09 nmol/10(9) cells) with a solanesol side chain was also identified.

Animals↗
PubMed1991

Californium-252 plasma desorption mass spectrometry of oligosaccharides and glycoconjugates: control of ionization and fragmentation.

The fragmentation of underivatized and peracetylated maltooligosaccharides, ranging in length from four to seven glucose residues, has been investigated by 252Cf plasma desorption mass spectrometry (PDMS). Investigations are made of the effects on the mass spectra of (1) peracetylation, (2) sample preparation by electrospray deposition onto metallic substrates versus adsorption onto nitrocellulose films, and (3) sodium addition to or elimination from the sample. Peracetylation enhances the mass spectrometric response of the oligosaccharides and also enables the efficient removal from the sample of water-soluble components such as sodium salts. A rapid and simple method has been developed for controlling the amount and type of fragmentation of peracetylated maltooligosaccharides. The method involves control of the amount of sodium in the sample introduced into the mass spectrometer. When a large molar excess of sodium is added to the sample, in the form of sodium chloride, the positive-ion mass spectrum is dominated by a peak corresponding to the sodium-cationized molecule. On the other hand, when the sample is completely depleted of sodium, the spectrum shows no quasimolecular ions [(M + Na)+ or (M + H)+] whatsoever and is instead totally dominated by fragment ion species. Thus, it proves feasible to alternate between a spectrum dominated by the (M + Na)+ ion peak and one dominated by fragment ions. This method has also been found to be useful in controlling the fragmentation of permethylated gangliosides.

Californium↗