NMR studies of substrate binding to cytochrome P450 BM3: comparisons to cytochrome P450 cam.

The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P450 BM3 (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 A. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 A from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P450 cam, and their implications for structural changes involved in the catalytic cycle are discussed.

The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide. NMR studies using selectively 15N-labelled protein.

The effects of the binding of the corepressor L-tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [15N]alanine, [15N]glutamate, [15N]isoleucine, [15N]leucine and [15N]methionine were prepared by isolating them from prototrophic E. coli cells grown in media containing a mixture of unlabelled and the appropriate 15N-enriched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues. All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the 'helix-turn-helix' DNA-binding domain, but also in residues in helices C and F remote from the ligand binding sites. On operator binding there is selective broadening of the signals of residues in the N-terminal region of the protein and in the DNA-binding domain, perhaps reflecting a conformational equilibrium.

Detection of foot-and-mouth disease antigen in bovine epithelial samples: comparison of sites of sample collection by an enzyme linked immunosorbent assay (ELISA) and complement fixation test.

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.

Infection of cattle by airborne foot-and-mouth disease virus: minimal doses with O1 and SAT 2 strains.

Equipment has been constructed and methods developed for exposing individual cattle to two strains of foot-and-mouth disease (FMD) virus in aerosols to determine the minimal infective dose by the respiratory route. The aerosols used were produced either artificially by a spinning-top aerosol generator, in which case they were of homogeneous small particle size (less than 3 micron in diameter) or else they were derived naturally from infected pigs, in which case the particles were heterogeneous in size. Two strains of FMD virus were used: an O1 strain of UK origin and a SAT 2 strain from South Africa. The lowest doses which initiated infection were 12.5 TCID50 of O1 BFS virus and 25 TCID50 of SAT 2 virus, infectivity having been assayed in primary bovine thyroid cell cultures. Following exposure to low doses of virus (range 12 to 316 TCID50) 33 per cent of the cattle exposed to O1 BFS virus and 27 per cent exposed to SAT 2 virus were infected but did not develop detectable vesicular lesions.

Exposure of sheep to natural aerosols of foot-and-mouth disease virus.

Sheep taken individually and allowed to inhale air being drawn along a duct from a cabinet containing pigs acutely infected with foot-and-mouth disease virus for 10 or 15 minute periods were infected by doses as low as 10 TCID50 of virus. The most consistent and reliable indicators of infection were viraemia and seroconversion. The mean times from exposure to onset of viraemia, pyrexia and the appearance of vesicular lesions were 2.5, 3.8 and 4.7 days, respectively. Neither the time from exposure to first detectable viraemia nor vesication correlated with dose. Around 27 per cent of sheep which were known to have been infected did not develop vesicles.

Choice of internal mammary artery or saphenous vein graft for myocardial revascularization.

The saphenous vein has been the traditional conduit for elective myocardial revascularization. Although readily available and adaptable to many configurations around the heart, it is prone to intimal hyperplasia and vein graft atherosclerosis, which diminish long-term patency and relief of symptoms. The internal mammary artery graft represents a marked improvement over the saphenous vein graft in many respects. Data are presented comparing saphenous vein graft patency with that of the internal mammary artery, bilateral internal mammary artery, free internal mammary artery, and sequential internal mammary artery grafts.

Response of sheep vaccinated with large doses of vaccine to challenge by airborne foot and mouth disease virus.

Administration of three-fold or six-fold larger doses of conventional monovalent type O foot and mouth disease (FMD) vaccine to sheep prevented viraemic distribution of virus after exposure to airborne virus one week later. However, virus replication in the respiratory tract or excretion in oesophageal-pharyngeal fluids and breath was not prevented. The implication of these findings for the use of vaccine as an adjunct to a 'stamping out' policy for countries which are free from FMD and which do not practice mass annual vaccination are discussed.