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Biomedical subjects

A Allerhand

Publications and source records attributed to A Allerhand.

At least 19 recordsLinked to original sources
PubMed1981

Kinetics of alpha-mannosidase action on various alpha-D-mannopyranosyl linkages in hen ovalbumin glycopeptides as monitored by carbon 13 nuclear magnetic resonance spectroscopy.

We show that 13C NMR spectroscopy is a practical method for following the kinetics of enzymatic digestion of individual carbohydrate residues of glycopeptides and for determining the structures of the products of partial digestions. Specifically, we study the jack bean alpha-mannosidase digestion of the two hen ovalbumin glycopeptides Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound A) and Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound B). The reported "rule" that jack bean alpha-mannosidase hydrolyzes Man alpha 1 leads to 2Man and Man alpha 1 leads to 6Man linkages at least 15 times faster than Man alpha 1 leads to 3Man linkages (Tai, T., Yamashita, K., Ogata-Arakawa, M., Koide, N., Muramatsu, T., Iwashita, S., Inoue, Y., and Kobata, A. (1975) J. Biol. Chem. 250, 8569-8575) is not of general validity. Although the Man alpha 1 leads to 2Man (alpha) linkage of Compound A is the first one to be digested, the Man alpha 1 leads to 3Man (beta) linkage is hydrolyzed next, faster than the Man alpha 1 leads to 6Man (alpha) linkage. The Man alpha 1 leads to 3Man (alpha) linkage is hydrolyzed very slowly. We show that it is practical to use 13C NMR spectroscopy to determine when the enzymatic digestion should be halted to isolate derivatives such as Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn and Man alpha 1 leads to 3Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn.

Animals↗
PubMed1981

Structure and dynamic behavior of the oligosaccharide side chain of bovine pancreatic ribonuclease B. Application of carbon 13 nuclear magnetic resonance spectroscopy.

Natural abundance 13C NMR (at 67.9 MHz) is used to study the primary structure and dynamic behavior of the carbohydrate side chain [(Man)6(GlcNAc)2-Asn] of ribonuclease B and of the shorter carbohydrate side chain [Man(GlcNAc)2-Asn] of a modified ribonuclease B (ribonuclease Bm). A comparison of the 13C NMR spectra of ribonuclease B and of the model compounds Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6 (Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound A) and Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc beta 1 leads to Asn (Compound B) indicates that the (Man)5(GlcNAc)2 configuration of Compound A is present as a core structure in ribonuclease B and that only up to about 30% of our sample of ribonuclease B has the (Man)6(GlcNAc)2 structure of Compound B. Spin-lattice relaxation times, nuclear Overhauser enhancements, and linewidths of the carbohydrate carbon resonances of ribonuclease Bm indicate that the mannose residue and the N-acetylglucosamine linked to mannose are undergoing fast internal rotation (at least as fast as the rate of overall molecular tumbling). The terminal mannose residues of ribonuclease B also exhibit fast internal rotation. A comparison of the chemical shifts of the nonprotonated aromatic carbons of ribonuclease B and ribonuclease A strongly suggests that the carbohydrate side chain of ribonuclease B has a negligible effect (overall or localized) on the conformation of bovine pancreatic ribonuclease.

Animals↗
PubMed1980

Tautomeric states of the histidine residues of bovine pancreatic ribonuclease A. Application of carbon 13 nuclear magnetic resonance spectroscopy.

Natural abundance 13C NMR (at 67.9 MHz) is used to study the nonprotonated aromatic carbons of bovine pancreatic ribonuclase A. The C gamma resonances of the 4 histidines are identified and assigned to specific residues. The effect of pH on the chemical shifts of these resonances yields information about the tautomeric state of the imidazole form of each histidine residue. Only His-119 exists predominantly or entirely in the "common" N epsilon 2-H (N tau-H) tautomeric state. His-12 and HIS-48 exist mainly or entirely in the N delta 1-H (N tau-H) tautomeric state. His-105 exhibits about 50 to 90% of the N delta 1-H tautomer. These results, when taken together with known crystallographic coordinates for ribonuclease A, strongly suggest that H delta 1 of His-105 is hydrogen bonded to the COOH-terminal carboxylate group, that H delta 1 of His-48 is hydrogen bonded to the carboxylate group of Asp-14 or (less likely) to the hydroxyl oxygen of Thr-17, and that H delta 1 of His-12 is probably hydrogen bonded to the hydroxyl oxygen of Thr-45.

Animals↗
PubMed1980

Natural abundance carbon 13 nuclear magnetic resonance spectroscopy of antifreeze glycoproteins.

Natural abundance 13C NMR spectroscopy (at 67.9 MHz) is used to study an aqueous solution of the antifreeze glycoproteins 3 to 6 from the Antarctic cod. Assignments of resonances to specific carbons of the known Ala-Ala-[beta Gal-(1 leads to 3)-alpha-N-acetylgalactosamine]Thr repeating unit are presented. Some of the carbons of the proline residues of glycoprotein 6 are also identified. Spin-lattice relaxation times (at 30 degrees C), linewidths (at 35 degrees C), nuclear Overhauser enhancements (at 35 degrees C), and the temperature dependence of chemical shifts (from 0 degrees to 75 degrees C) all strongly suggest that these antifreeze glycoproteins are predominantly or entirely flexible random coil polymers.

Amino Acids↗
PubMed1979

Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate.

It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).

Egg White↗
PubMed1977

Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence.

The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.

Amino Acid Sequence↗
PubMed1977

Participation of tryptophan 62 in the self-association of hen egg white lysozyme. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Self-association of hen egg white lysozyme in solution of 38 degrees) is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. The effect of pH on the resonances of the nonprotonated aromatic carbons of 9 mM lysozyme, and the effect of protein concentration (at pH 7) on these resonances, both indicate that self-association significantly affects the chemical shift of Cgamma of Trp-62, but not the chemical shifts of the other nonprotonated aromatic carbons. This result is consistent with the reported participation of Trp-62 in the intermolecular contact (Banerjee, S.K., Pogolotti, A., and Rupley, J.A. (1975) J. Biol. Chem. 250, 8260-8266). Our results indicate that the resonance of Cgamma or Trp-62 is a convenient monitor of lysozyme self-association. The chemical shift of this resonance reflects the extent of aggregation, while the line width yields information about the lifetime of the intermolecular contact. This lifetime is 1 to 2 ms at 38 degrees (9 mM protein, 0.1 M NaCl, pH 7). Our results also indicate that self-association of lysozyme is not accompanied by any general conformational change, and that binding of a lanthanide ion (at the metal ion binding site near the carboxylate groups of ASP-52 AND Glu-35) strongly suppresses self-association.

Animals↗
PubMed1977

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

Azurin↗