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Biomedical subjects

N C Price

Publications and source records attributed to N C Price.

159 records · Page 9Linked to original sources
PubMed1975

Antibody--hapten interactions in solution.

This paper reports the initial progress in a research programme to identify and obtain the relative orientations, in solution, of the amino acid residues that constitute the combining site of the myeloma protein MOPC 315. This protein has a molecular mass of 150,000, but enzymic digestion yields the Fv fragment of molecular mass 25,000 which still has the combining site intact, as judged by the affinity for dinitrophenyl haptens. Analysis of the e.s.r. spectra of a series of dinitrophenyl spin labelled haptens has allowed the dimensions, rigidity and polarity profile of the combining site to be determined. The combining site is a cleft of overall dimensions 1.1 nm x 0.9 nm x 0.6 nm which has considerable structural rigidity. One of these spin labels has also been used to perturb the n.m.r. spectrum of the Fv and using difference spectroscopy the 270 MHz proton n.m.r. spectrum of the amino acid residues in and around the combining site has been obtained. This spectrum contains only the equivalent of about 30 aromatic and 21 aliphatic protons. Comparison of this difference spectrum with that obtained using a diamagnetic analogue suggests that any conformational changes on hapten binding are mainly localized to the combining site. By the use of (n.m.r.) difference spectroscopy the protons of the three histidine residues in the Fv are observed to titrate with pH and have pKa values of about 8.1, 6.9 and 6.1. The histidine resonances with pKa values 6.9 and 6.1 alter slightly in the presence of haptens and also appear in the spin label difference spectrum, and must therefore be in or near to the combining site. These are assigned to His 102H and His 97L. The existence of lanthanide binding sites on the Fv, necessary for the mapping studies, has been demonstrated by measurements of Gd III water relaxation rates in Fv solutions and also by the changes in the Fv tryptophan fluorescence on addition of Gd III. At pH 5.5 there is one tight binding site for the lanthanides (KD approximately 80 muM) but in the presence of hapten this is weakened 10-20 fold with a reciprocal effect on the hapten binding. Measurements of the Gd III quenching of the e.s.r. spectrum of a spin labelled hapten bound to Fv indicate that the lanthanide site is ca. 1.5 nm from the nitroxide moiety.

Amino Acids↗
PubMed1975

Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase.

The environments of the two sulfhydryl groups of procine muscle adenylate kinase have been investigated by chemical modification reactions. The results indicate that the environments of the two-SH groups of procine muscle adenylate kinase are markedly different and that substrates induce conformational changes in the enzyme in the region of the sulfhydryl groups. The fluorogenic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-chloride) reacts specifically with the -SH groups of the enzyme at pH 7.9. One thiol group reacts with NBD-chloride approximately 40-fold faster than the other one, and the fast reacting group has been identified as Cys-25 in the amino acid sequence. The similarity of the rate of the more slowly reacting Cys-187 with NBD-chloride to that of glutathione with the same reagent is consistent with its location on the surface of the enzyme as determined by x-ray crystallography structure. The fast reacting Cys-25 in the interior of the structure can be approached by compounds such as NBD-chloride via a cleft. Reaction of Cys-25, presumably located close to the catalytic center, leads to complete inactivation of the enzyme. Substrates such as ATP, MgATP, and ADP which bind to the triphosphate subsite of the enzyme decrease the rate of reaction of Cys-25 by factors up to 3.5 but have only a small effect (approximately equal to 10%) on the reactivity of Cys-187. AMP, however, has a pronounced effect on the reactivity of Cys-187, the slowly reacting group. The multisubstrate analogue P-1, P-5-di-(adenosine-5)pentaphosphate (Ap-5A) decreases the rate of reaction of the fast reacting thiol group by a factor of 300. The behavior of Cys-25 toward NBD-chloride, i.e. super-reactivity in the absense of Ap-5A and slow reactivity in the presence of the multisubstrate inhibitor, was characteristic for both porcin and carp adenylate kinase. In the presence of Ap-5A adenylate kinase can be selectively modified at Cys-187; the introduction of the fluorescent NBD group at this position has no effect on enzymatic activity. A slow transfer of the NBD group occurs from the third groups to the epsilon-amino group of Lys-31. This transfer reaction is further evidence that the structure of adenylate kinase in dilute solution is similar to that of the crystalline enzyme since the x-ray data have shown that the sulfur of Cys-187 and the epsilon-nitrogen of Lys-31 are less than 4 A apart. The strongly fluorescent NBD-NH-enzyme possesses full activity and binds substrates as. cont'd

Adenosine Monophosphate↗
PubMed1975

A kinetic method to establish the specificity of spin labelling of macromolecules.

A method has been devised for establishing the specificity of spin labelling of a macromolecule. Analysis of the rate of incorporation of spin label under different conditions permits the assignment of reacting groups to classes of differing reactivity. The method is illustrated for the enzyme glycogen phosphorylase b.

Electron Spin Resonance Spectroscopy↗
PubMed1969

Desensitization of glutamate dehydrogenase by reaction of tyrosne residues.

1. The reaction of glutamate dehydrogenase with N-acetylimidazole and with tetranitromethane leads to modification of tyrosine residues. 2. Modification of 1 tyrosine residue/subunit does not affect the enzymic activity but decreases the response of the enzyme to the allosteric inhibitor, GTP. 3. The physical properties of the enzyme (sedimentation coefficient and optical rotatory dispersion) remain unaltered. 4. GTP partially protects against desensitization. 5. The diminished responses of the modified enzymes to GTP are also detected by using the fluorescence of 1-anilinonaphthalene-8-sulphonate as a conformational probe. 6. Difficulties that generally arise in chemical modifications from inhomogeneous distributions of products are discussed.

Aniline Compounds↗