Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases.

The family of glutamate dehydrogenases include a group of hexameric oligomers with a subunit M(r) of around 50,000, which are closely related in amino acid sequence and a smaller group of tetrameric oligomers based on a much larger subunit with M(r) 115,000. Sequence comparisons have indicated a low level of similarity between the C-terminal portion of the tetrameric enzymes and a substantial region of the polypeptide chain for the more widespread hexameric glutamate dehydrogenases. In the light of the solution of the three-dimensional structure of the hexameric NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequence for the C-terminal domain of the tetrameric Neurospora crassa glutamate dehydrogenase against the sequence and the molecular structure of that from C. symbiosum. This analysis reveals that the residues conserved between these two families are clustered in the three-dimensional structure and points to a remarkably similar layout of the glutamate-binding site and the active-site pocket, though with some differences in the mode of recognition of the nucleotide cofactor.     

Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum. Evidence for a ligand-induced conformational change.

A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion. The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate. The crystals diffract well and X-ray photographs have established that they are in the space group R32. Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit. Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell. Such a conformational rearrangement may represent an important step in the catalytic cycle.     

Fire and the chemistry of a South African mountain stream

The effects of a late-summer prescribed burn on the chemistry of a second-order mountain stream in the south-western Cape, South Africa, were investigated. Nitrate concentrations in stream water were significantly higher during the winter of the post-burn year. Increased concentrations of chloride, bicarbonate, polyphenols and potassium and decreased sodium concentrations were also recorded. Concentrations of ammonium, phosphate, calcium, magnesium, total dissolved solids and hydrogen ions were not significantly affected by the burn. Ionic export from the catchment was generally greater in the post-burn year. Apart from nitrate, however, values probably lie within the natural range of year-to-year variation. It is predicted that enhanced losses of nitrate will decrease progressively with the recovery of the vegetation and the re-establishment of soil/plant nutrient cycles. Atmospheric losses of nutrients in smoke were unquantified, but may be of more significance to site productivity than losses through surface runoff, which, in the case of nitrogen, appear to be compensated by precipitation inputs.     

Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an‘up-regulated’promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.     

Studies of Photosynthesis Using a Pulsed Laser: I. Temperature Dependence of Cytochrome Oxidation Rate in Chromatium. Evidence for Tunneling

The rate of oxidation of cytochrome following absorption of a short pulse of light from a ruby laser in the photosynthetic bacterium Chromatium has been measured spectrophotometrically. The half-time is about 2 musec at room temperature increasing to 2.3 msec at about 100 degrees K and constant at the latter value to 35 degrees K or below. The temperature dependence above 120 degrees K corresponds to an activation energy of 3.3 kcal/mole; that below 100 degrees K to less than 80 cal/mol: essentially a temperature-independent electron transport reaction. Since the slowness below 100 degrees K indicates the presence of a barrier, the lack of activation energy is taken to mean penetration by quantum-mechanical "tunneling."     

The biosynthesis of cyclic carotenes

1. The incorporation of (3RS)-[2-(14)C,(4R)-4-(3)H(1)]mevalonic acid into various cyclic carotenes in the fruit of the tomato mutant delta has been studied. The results confirm our previous view that the alpha-ionone ring of alpha-carotene does not arise by isomerization of a beta-ionone residue, and show that the same is also true for the alpha-ionone ring of delta- and in-carotene and alpha-zeacarotene. 2. The incorporation of (3RS)-[2-(14)C,2-(3)H(2)]mevalonic acid into alpha- and beta-carotene in carrot roots has been studied. The results show that the beta-ionone ring of beta-carotene does not arise by isomerization of the alpha-ionone residue of alpha-carotene. 3. These experiments show that alpha- and beta-ionone rings in cyclic carotenes are formed independently, probably by elimination of different protons from the same carbonium ion intermediates.     

NAD(P) redox compartmentation in Ehrlich ascites tumour cells

1.

1. Normoxia-anoxia transitions and use of glycolytic inhibitors and substrates have made possible a distinction between NAD(P)H redox changes in the mitochondrial and cytosolic compartments of ELD (Ehrlich-Lettré hyperdiploid) ascites tumour cells.     

Biosynthesis of 1,2-dihydrocarotenoids in Rhodopseudomonas viridis: Experiments with inhibitors

The effects of the inhibitors diphenylamine (DPA), 2-(4-chlorophenylthio) triethylammonium chloride (CPTA) and nicotine on the biosynthesis of 1,2-dihydrocarotenoids by Rhodopseudomonas viridis (Rhodospirillaceae) have been investigated. Small amounts of 1,2-dihydro derivatives of phytoene, phytofluene and ξ-carotene and its unsymmetrical isomer, and 1,2,1′,2′,-tetrahydro derivatives of neurosporene and lycopene were isolated from R. viridis grown in the presence of DPA, although there was virtually no quantitative effect on the levels of the normal main carotenoids, neurosporene and lycopene and their 1,2-dihydro derivatives. Nicotine also had little effect on the overall carotenoid composition, but the formation of 1,2-dihydrocarotenoids was inhibited to some extent by CPTA. The 1,2-dihydro end group may thus be introduced by a hydrogenation reaction similar to the more familiar C-1,2 hydration reaction characteristic of carotenoid biosynthesis in other photo synthetic bacteria.     

Colonoscopy in surgical practice.

Colonoscopy is a rewarding new technique with a potential for early and more accurate diagnosis. One hundred and seventy colonoscopies carried out over the past three years showed or confirmed colonic cancer in 14 patients, and solitary or multiple colonic polyps were found in 28 cases, of which 18 were excised endoscopically. A large villous adenoma was diagnosed in one patient, and the absence of a suspected sinister lesion was shown by direct examination and biopsy in 110 cases. There were 17 examination failures, including two perforations of the bowel. Colonoscopy complements rather than supplants barium enema examination and will make diagnostic laparotomy for colonic lesions unnecessary. The use of the diathermy snare allows endoscopic removal of colonic polyps and should greatly reduce the need for formal surgery in these cases. The financial saving to the Health Service will greatly outweigh the expense of the procedure, but it should be undertaken only in well organised centres as a specialist service for selected patients. In skilled hands it is safe, but potential hazards exist for the inexperienced endoscopist.     

The effect of illumination on the pigment composition of the ζ-carotenic mutant,PG1, of Scenedesmus obliquus

Dark grown Scenedesmus obliquus strain PG1 accumulated -carotene and phytoene as its major carotenoids. On illumination of dark-grown cultures in air/CO₂, cyclic carotenes and xanthophylls were formed, apparently at the expense of the accumulated phytoene and -carotene. This interconversion of carotenoids was accompanied by chlorophyll synthesis. In an atmosphere of nitrogen/CO₂ the light-induced changes occurred more slowly and in nitrogen alone the changes were incomplete. No massive production of cyclic carotenes from the accumulated -carotene was observed in cultures illuminated under anaerobic conditions.