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K Biemann

Publications and source records attributed to K Biemann.

At least 91 records · Page 5Linked to original sources
PubMed1984

Strategy for the mass spectrometric verification and correction of the primary structures of proteins deduced from their DNA sequences.

Fast atom bombardment mass spectrometry has been used to confirm and correct regions from the amino acid sequences of three large proteins, glutaminyl- and glycyl-tRNA synthetase from Escherichia coli and methionyl-tRNA synthetase from yeast, whose primary structures had been deduced from the base sequences of their corresponding genes. The strategy is based on a comparison of the molecular weights of the tryptic peptides predicted from all three reading frames of the gene sequences with those determined mass spectrometrically. The experimental molecular weights either match or differ and can be used to assess the correctness of the base sequences, identify errors that lead to frame shifts, premature stop codons, incorrect amino acids, etc., or identify the presence of posttranslational modifications. This method is very fast and requires little material (5-20 nmol).

Amino Acid Sequence↗
PubMed1983

Primary structures of both subunits of Escherichia coli glycyl-tRNA synthetase.

Escherichia coli glycyl-tRNA synthetase is one of two aminoacyl-tRNA synthetases which is comprised of two different subunits (in an alpha 2 beta 2 structure). The two coding regions occur in tandem in the order alpha + beta and are synthesized from a single mRNA (Keng, T., Webster, T. A., Sauer, R. T., and Schimmel, P. R. (1982) J. Biol. Chem. 257, 12503-12508). Primary structures of both proteins were determined by DNA sequencing of each coding region and by analysis of tryptic fragments of the enzyme. The alpha-subunit is 303 codons and terminates with TAA; the beta-subunit is 689 codons followed by tandem TAA stops. S1 nuclease mapping of the 3'-end of the two-cistron glyS mRNA showed that it predominantly ends 33/34 bases beyond the tandem stops with an RNA polymerse terminator sequence. Altogether, 43% of the translated polypeptide sequences were confirmed by mass spectrometric analysis of peptide fragments including confirmation of the COOH-terminal end of the beta-chain. This involved determinations, by fast atom bombardment mass spectrometry, of the masses of numerous whole tryptic fragments (with an accuracy of better than 1 Da) and of fragments truncated by one to three cycles of Edman degradations. The primary structures of the two subunits show no homologies with each other and have no internal sequence repeats of significance. While there are no extensive homologies with five other sequenced, or partially sequenced, synthetases, the alpha-subunit has a short sequence which can be aligned with sequences found in functionally important areas of two other synthetases and in uncharacterized parts of a third and fourth synthetase.

Amino Acid Sequence↗
PubMed1983

Primary structure of macromomycin, an antitumor antibiotic protein.

The antitumor protein macromomycin is a single chain polypeptide of 112 amino acid residues cross-linked by two intramolecular disulfide bonds. The protein was reduced and S-alkylated with 2-mercaptoethanol in 8 M urea followed by treatment with iodoacetic acid. Tryptic digestion of tetra-S-carboxymethyl macromomycin gave four tryptic peptides which were fractionated by gel permeation on Sephadex G-50. The amino acid sequence of the tryptic peptides and the overlap sequences were determined by a combination of automated Edman degradation analysis, gas chromatographic mass spectrometry, and fast atom bombardment mass spectrometry. A comparison of the structures of macromomycin, actinoxanthin, and neocarzinostatin suggests that they belong to a family of related proteins.

Amino Acid Sequence↗
PubMed1983

Confirmation of the primary structure of thymosin alpha1 by microsequence analysis of limited acid and enzymatic hydrolysis fragments.

The primary structure of the 28-peptide thymosin alpha 1 as determined by Goldstein et al. (1) has been confirmed by independent procedures. Limited dilute acid digestion generated a 26-peptide and a 22-peptide both extending to the C-terminal and lacking the N-terminal blocking group. A combination of Edman microsequencing, carboxypeptidase Y and thermolysin digestion, and fast atom bombardment mass spectrometry was used.

Amino Acid Sequence↗
PubMed1982

Escherichia coli glutaminyl-tRNA synthetase. II. Characterization of the glnS gene product.

Glutaminyl-tRNA synthetase has been purified by a simple, two-column procedure from an Escherichia coli K12 strain carrying the glnS structural gene on plasmid pBR322. The primary sequence of this enzyme as derived from the DNA sequence (see accompanying paper) has been confirmed. Manual Edman degradation was used to identify the NH2-terminal sequence of the protein. Oligopeptides scattered throughout the primary sequence of glutaminyl-tRNA synthetase were sequenced by the gas chromatographic-mass spectrometric method and matched to the theoretical peptides derived from the translated DNA sequence. The expected carboxyl terminus at position 550 was verified by carboxypeptidase B digestion. The primary sequence of glutaminyl-tRNA synthetase contains no extensive sequence repeats. A search was made for sequence homologies between this enzyme and the few other aminoacyl-tRNA synthetases for which primary sequences are available. A single homologous region is shared by at least three of the synthetases examined here.

Amino Acid Sequence↗
PubMed1982

The site of attachment of retinal in bacteriorhodopsin. The epsilon-amino group in Lys-41 is not required for proton translocation.

Chymotryptic fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71) of bacteriorhodopsin have been shown previously to reassociate so as to regenerate the native bacteriorhodopsin chromophore in lipid/detergent mixtures and to form functional proton-translocating vesicles. The fragment C-2 has now been selectively methylated with formaldehyde and sodium cyanoborohydride to give the epsilon-dimethylamino derivatives of Lys-30, 40, and 41 in 96-99% average yield. The methylated and unmethylated C-2 fragments were identical in their ability to reassociate with fragment C-1 and retinal to regenerate the bacteriorhodopsin chromophore and to form functional proton-translocating vesicles. In contrast, dimethylation of the lysine residues of the C-1 fragment gave a derivative which did not form an active complex with unmethylated C-2. We conclude that the epsilon-amino group in Lys-41 is not required for Schiff's base formation with retinal at any step in the light-driven proton-translocation cycle.

Bacteriorhodopsins↗
PubMed1982

Primary structure of monkey osteocalcin.

The complete 49-residue amino acid sequence of osteocalcin from the old world monkey Macaca fascicularis has been determined by efficient combination of gas chromatography-mass spectrometry and Edman techniques. This vitamin K dependent protein of bone matrix contains three gamma-carboxyglutamic acid residues at positions 17, 21, and 24, as well as a disulfide-bonded loop (23--29). Features of the sequence which apparently are required for the binding of Ca2+ have been strongly conserved throughout evolution.

Amino Acid Sequence↗
PubMed1982

Simultaneous determination of phenobarbital and p-hydroxyphenobarbital and their stable isotope labeled analogs by gas chromatography mass spectrometry.

A method suitable for pulse dosing studies is described for the quantitation by gas chromatography mass spectrometry of phenobarbital (5-ethyl-5-phenylbarbituric acid), p-hydroxyphenobarbital (5-ethyl-5(4-hydroxyphenyl)barbituric acid) and, simultaneously, their (13C15N2)-labeled analogs in serum and urine. Differently labeled analogs are used as internal standards (5-ethyl-5(2,3,4,5-tetradeuterophenyl)-2-(13C)barbituric acid for quantitation of phenobarbital, and 5-ethyl-5(4-hydroxy-3,5-dideuterophenyl)2-(13C)-1,3-(15N2)barbituric acid for p-hydroxyphenobarbital). In the procedure, the chemical work-up of the samples (1.0 ml for serum, 0.5 ml for urine) is based on an extractive methylation technique for the generation of the permethylated derivatives. The mass spectrometric measurement technique consists of repetitive scanning over a preselected mass region of the permethylated derivatives of the analytes as they elute from the gas chromatograph. The method is evaluated for serum concentrations ranging from 0.1-30.0 micrograms ml-1 for phenobarbital and 0.1-10.0 micrograms ml-1 for p-hydroxyphenobarbital, and for urine concentrations of both phenobarbital and p-hydroxyphenobarbital of 1.0-50.0 micrograms ml-1. Application of the method to determination of the pharmacological equivalence of phenobarbital and (13C15N2)-labeled phenobarbital in man is also demonstrated.

Carbon Isotopes↗
PubMed1982

Pharmacokinetic equivalence of stable-isotope-labeled and unlabeled drugs. Phenobarbital in man.

Stable isotope labeling of drugs has been used in human metabolism studies because it eliminates the risk of radiation exposure accompanying use of radioactive tracers. The labeled drug can be measured by gas-chromatographic mass spectrometry (GCMS). However, if reliable pharmacokinetic data are to be obtained, one has to be certain the rate of metabolism of labeled and unlabeled drug is the same, i.e., there is no kinetic isotope effect. To evaluate this for phenobarbital (PB), three humans were infused with a 1:1 mixture of phenobarbital and 1,3-15N2-2-13C-PB. Serum was collected at regular intervals. Concentrations of labeled and unlabeled phenobarbital were determined by GCMS. Within each subject, there was no trend for concentrations of labeled phenobarbital to be higher or lower than concentrations of unlabeled phenobarbital (P greater than 0.90 for all three subjects). There was no difference in the zero time intercepts, distribution and elimination time constants and half-lives, volumes of distribution and central compartment, or clearance of the two forms of phenobarbital. Thus, no isotope effect was found. Published data on other labeled drugs and the likelihood of encountering an isotope effect based on type of isotope and its location in the molecule are discussed.

Adult↗
PubMed1982

n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle.

The unusual NH2-terminal blocking group of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein was found to be amide-linked n-tetradecanoic acid by gas chromatographic-, direct chemical ionization-, and fast atom bombardment-mass spectrometry. In addition, fast atom bombardment mass spectrometry revealed the presence of an additional alanine which had been overlooked when the original sequence was determined. The corrected and completed NH2-terminal sequence of the 350-amino acid catalytic subunit is CH3(CH2)12CONH-Gly-Asn-Ala-Ala-Ala-Ala-Lys.

Amino Acid Sequence↗
PubMed1982

Chemical and in vivo studies of the anion oxo[N,N'-ethylenebis(2-mercaptoacetimido)]Technetate(V).

Studies of the anionic coordination complex 99Tc-oxo[N,N'-ethylene-bis(2-mercaptoacetimido)]technetate(V) ([TcO(ema)]-) are described. Syntheses performed both at carrier levels (10(-5)M) and with no carrier added (less than 10(-8)M) indicate that the complex is formed virtually quantitatively from pertechnetate ion over this range. Tissue distributions in normal rats are similar at both concentrations up to one hour after administration. It has been shown--using a combination of high-pressure liquid chromatography and field-desorption mass spectrometry--that the anion is excreted unchanged into both urine and bile. The effectiveness of this N2S2 donor set in sequestering Tc-99m, and the in vivo stability of the resulting complex, suggest that modified chelates of this structural class could provide a series of useful diagnostic agents.

Animals↗
PubMed1981

Gas chromatographic mass spectrometric sequence determination of osteocalcin, a gamma-carboxyglutamic acid-containing protein from chicken bone.

The complete primary structure of osteocalcin, the gamma-carboxyglutamic acid (Gla)-containing calcium-binding protein isolated from chicken bone has been determined by gas chromatographic mass spectrometry. The method involves decarboxylation of the dry, intact protein in a low pressure atmosphere of DCl under conditions which quantitatively convert Gla into gamma, gamma-dideuteroglutamic acid residues without resulting in peptide bond cleavage. After partial enzymatic or acidic hydrolysis, the peptide mixtures are converted (without isolation of individual peptides) to the N-trifluorodideuteroethyl O-trimethylsilyl polyamino alcohols and analyzed by gas chromatographic mass spectrometry. Peptide fragments containing former Gla residues behave like Glu-containing peptides, but their mass spectra are shifted upward by 2 mass units/Gla residue. Chicken osteocalcin contains 50 amino acids, three of which are Gla. The structure is highly homologous to the sequence of the corresponding protein isolated from bovine bone, and the relative sequence locations of the Gla residues and the disulfide bridge are conserved.

1-Carboxyglutamic Acid↗