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Biomedical subjects

R N Perham

Publications and source records attributed to R N Perham.

At least 163 records · Page 9Linked to original sources
PubMed1981

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

Amino Acid Sequence↗
PubMed1981

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complexes from bacterial and mammalian sources.

A simple method was developed for assessing the intramolecular coupling of active sites in the lipoate acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complexes from Escherichia coli, Bacillus stearothermophilus and ox heart and pig heart mitochondria. Samples of enzyme complex were prepared in which the pyruvate decarboxylase (E1) component was selectively and partly inhibited by treatment with increasing amounts of a transition-state analogue, thiamin thio-thiazolone pyrophosphate. The fraction of the E2 component acetylated by incubation with [2-14C] pyruvate, in the absence of CoA, was determined for each sample of partly inhibited enzyme and was found in all cases to exceed the fraction of overall complex activity remaining. This indicated the potential for transacetylation reactions among the lipoic acid residues within the E2 core. A graphic presentation of the data allowed comparison of the active-site coupling in the various enzymes, which may differ in their lipoic acid content (one or two residues per E2 chain). It is clear that active-site coupling is a general property of pyruvate dehydrogenase complexes of octahedral and icosahedral symmetries, the large numbers of subunits in each E2 core enhancing the effect.

Acetylation↗
PubMed1980

Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.

A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.

Animals↗
PubMed1980

Purificaton of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and resolution of its four component polypeptides.

1. The pyruvate dehydrogenase complex was purified from Bacillus stearothermophilus in high yield. The specific activity (about 40nkat/mg of protein) was substantially lower than that of the pyruvate dehydrogenase complex from Escherchia coli (about 570nkat/mg of protein) measured at 30 degrees C under the same conditions. 2. The relative molecular masses of the four types of polypeptide chain i the complex were estimated by means of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to be 57 000, 54 000, 42 000 and 36 000 respectively. These polypetide chains showed no evidence of seriously anomalous behavior during tests of electrophoretic mobility. 3. The enzyme complex was resolved into its constituent proteins by means of gelfiltration on Sepharose CL-6B in the presence of 2M-KI, followed by chromatography on hydroxyapatite in the presence of 8M-urea. These harsh conditions were necessary to cause suitable dissociation of the enzyme complex. 4. The amino-acid compositions of the four constituent proteins after resolution were determined and their chain ratios were measured for several preparations of the complex. Some variability was noted between preparations but all samples contained a significant molar excess of the chains thought to contribute the pyruvate decarboxylase (EC 1.2.4.1) activity. 5. From the relative molecular masses and chain ratios of the four constituent proteins, it was calculated that the empirical unit must be repeated at least 50 times to make up the assembled complex. This conclusion is fully consistent with the demonstration by means of electron microscopy of apparent icosahedral symmetry for the Bacillus stearothermophilus complex, implying a 60-fold repeat. The structure stands in sharp contrast with the octahedral symmetry (24-fold repeat) of the Escherichia coli enzyme.

Amino Acids↗
PubMed1980

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.

Escherichia coli↗
PubMed1979

Extended amino acid sequences around the active-site lysine residue of class-I fructose 1,6-bisphosphate aldolases from rabbit muscle, sturgeon muscle, trout muscle and ox liver.

1. Amino acid sequences covering the region between residues 173 and 248 [adopting the numbering system proposed by Lai, Nakai & Chang (1974) Science 183, 1204-1206] were derived for trout (Salmo trutta) muscle aldolase and for ox liver aldolase. A comparable sequence was derived for residues 180-248 of sturgeon (Acipenser transmontanus) muscle aldolase. The close homology with the rabbit muscle enzyme was used to align the peptides of the other aldolases from which the sequences were derived. The results also allowed a partial sequence for the N-terminal 39 residues for the ox liver enzyme to be deduced. 2. In the light of the strong homology evinced for these enzymes, a re-investigation of the amino acid sequence of rabbit muscle aldolase between residues 181 and 185 was undertaken. This indicated the presence of a hitherto unsuspected -Ile-Val-sequence between residues 181 and 182 and the need to invert the sequence -Glu-Val- to -Val-Glx- at positions 184 and 185. 3. Comparison of the available amino acid sequences of these enzymes suggested an early evolutionary divergence of the genes for muscle and liver aldolases. It was also consistent with other evidence that the central region of the primary structure of these enzymes (which includes the active-site lysine-227) forms part of a conserved folding domain in the protein subunit. 4. Detailed evidence for the amino acid sequences proposed has been deposited as Suy Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Amino Acid Sequence↗
PubMed1979

Effects of modification of the tyrosine residues of bacteriorhodopsin with tetranitromethane.

Treatment of the purple membrane of Halobacterium halobium with tetranitromethane led to modification of tyrosine residues. Modification of more than 3-4 tyrosine residues per bacteriorhodopsin monomer caused a decrease in the light-induced proton-pumping ability of purple membrane in synthetic lipid vesicles, loss of the sharp X-ray-diffraction patterns characteristic of the crystal lattice, loss of the absorbance maximum at 560 nm, and change in the buoyant density of the membrane. No modification of lipid was detected. These changes were interpreted as a gradual denaturation of the protein component such that when 8-9 tyrosine residues are modified, no proton pumping is observed. Modification of less than 3-4 tyrosine residues with tetranitromethane caused an increse in light-induced proton pumping. It was possible to generate partly modified purple membrane which had completely lost the property of diffracting X-rays into the sharp pattern observed with native purple membrane, but which still retained the ability to pump protons in a vectorial manner. Retention of crystal lattice is not essential for proton pumping.

Bacteriorhodopsins↗
PubMed1979

Polypeptide-chain stoicheiometry and lipoic acid content of the pyruvate dehydrogenase complex of Escherichia coli.

The pyruvate dehydrogenase multienzyme complex was isolated from Escherichia coli grown in the presence of [35S]sulphate. The three component enzymes were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the molar ratios of the three polypeptide chains were determined by measurement of the radioactivity in each band. The chain ratio of lipoamide dehydrogenase to lipoate acetyltransferase approached unity, but there was a molar excess of chains of the pyruvate decarboxylase component. The 35S-labelled complex was also used in a new determination of the total lipoic acid content. It was found that each polypeptide chain of the lipoate acetyltransferase component appears to bear at least three lipoyl groups.

Chemical Phenomena↗