Reduction of selenite and detoxification of elemental selenium by the phototrophic bacterium Rhodospirillum rubrum.

The effect of selenite on growth kinetics, the ability of cultures to reduce selenite, and the mechanism of detoxification of selenium were investigated by using Rhodospirillum rubrum. Anoxic photosynthetic cultures were able to completely reduce as much as 1. 5 mM selenite, whereas in aerobic cultures a 0.5 mM selenite concentration was only reduced to about 0.375 mM. The presence of selenite in the culture medium strongly affected cell division. In the presence of a selenite concentration of 1.5 mM cultures reached final cell densities that were only about 15% of the control final cell density. The cell density remained nearly constant during the stationary phase for all of the selenite concentrations tested, showing that the cells were not severely damaged by the presence of selenite or elemental selenium. Particles containing elemental selenium were observed in the cytoplasm, which led to an increase in the buoyant density of the cells. Interestingly, the change in the buoyant density was reversed after selenite reduction was complete; the buoyant density of the cells returned to the buoyant density of the control cells. This demonstrated that R. rubrum expels elemental selenium across the plasma membrane and the cell wall. Accordingly, electron-dense particles were more numerous in the cells during the reduction phase than after the reduction phase.

Outcome of clinical foot examination in relation to self-perceived health and glycaemic control in a group of urban Tanzanian diabetic patients.

Diabetic foot complications were studied in 153 patients at the university clinic in Dar es Salaam (56 insulin treated, 77 treated with oral agents and 20 with diet only). Neuropathy disability and symptoms scores were used to diagnose peripheral neuropathy (PN). Peripheral vascular disease (PVD) was classified as ankle/brachial pressure index less than one. The degree of metabolic control was assessed by glycated haemoglobin (HbA1c) and self-perceived health was measured with SF-36. PN was present in 28.1% of patients and 12.5% had PVD. Patients with PN had higher age and later onset of diabetes in comparison with patients without food complications. Patients with PVD had longer duration of diabetes and higher systolic and diastolic blood pressure compared with those free from PVD. HBA1c and body mass index did not seem to influence the occurrence of PN or PVD. Patients with PN had significantly poorer self-perceived health, whilst PVD-patients had health scorings equal to patients without any foot complications. PN, but not PVD, appeared to have a negative influence on patients self-perceived health. In comparison with studies from the industrial world, foot problems are as common in diabetic patients living in a developing country.

Purine nucleoside phosphorylase. 1. Structure-function studies.

To probe the catalytic mechanism of human purine nucleoside phosphorylase (PNP), 13 active-site mutants were constructed and characterized by steady-state kinetics. In addition, microtiter plate assays were developed for both the phosphorolytic and synthetic reactions and used to determine the kinetic parameters of each mutant. Mutations in the purine binding site exhibited the largest effects on enzymatic activity with the Asn243Ala mutant resulting in a 1000-fold decrease in the kcat for inosine phosphorolysis. This result in combination with the crystallographic location of the Asn243 side chain suggested a potential transition state (TS) structure involving hydrogen bond donation by the carboxamido group of Asn243 to N7 of the purine base. Analogous to the oxyanion hole of serine proteases, this hydrogen bond was predicted to aid catalysis by preferentially stabilizing the TS as a consequence of the increase in negative charge on N7 that occurs during glycosidic bond cleavage and the associated increase in the N7-Asn243 hydrogen bond strength. Two residues in the phosphate binding site, namely His86 and Glu89, were also predicted to be catalytically important based on their alignment with phosphate in the X-ray structure and the 10-25-fold reduction in catalytic activity for the His86Ala and Glu89Ala mutants. In contrast, catalytic efficiencies for the Tyr88Phe and Lys244Ala mutants were comparable with wild-type, indicating that the hydrogen bonds predicted in the initial X-ray structure of PNP [Ealick, S. E., et al. (1990) J. Biol. Chem. 265, 1812-1820] were not essential for catalysis. These results provided the foundation for studies reported in the ensuing two manuscripts focused on the PNP catalytic mechanism [Erion, M. D., et al. (1997) Biochemistry 36, 11735-11748] and the use of mutagenesis to reverse the PNP substrate specificity from 6-oxopurines to 6-aminopurines [Stoeckler, J. D., et al. (1997) Biochemistry 36, 11749-11756].

Purification of an LHI-RC-complex of Rhodospirillum rubrum by solubilization of chromatophores with a short-chain lecithin.

Chromatophores from Rhodospirillum rubrum were solubilized using the detergent 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC). The solubilization curves are sigmoidal reaching a plateau at a detergent/protein ratio of 2-3 µmol/mg corresponding to 75-90% solubilized protein. The BChl-binding proteins are stable over a large range of DHPC/protein ratios. A complex of BChl-binding proteins containing both LHI- and RC-polypeptides (LHI-RC-complex) was purified using a two step procedure. RC photochemical activity as well as absorption and near-IR CD spectra showed the complex to be active and stable after purification in presence of DHPC.

Short-chain phosphatidylcholines as superior detergents in solubilizing membrane proteins and preserving biological activity.

The solubilization of plasma and organelle membranes by diheptanoylphosphatidylcholine (DHPC) has been studied. This short-chain phosphatidylcholine is shown to act as a mild detergent, solubilizing effectively both kinds of membranes at DHPC concentrations of 10-20 mM (0.5-1%). The size of the resulting mixed protein-lipid-DHPC micelles ranges between 5 and 8 nm. The protein conformation and hence the enzymatic activity are well preserved over a rather large DHPC concentration range (up to 4-5 times the DHPC concentration required for solubilizing the membranes). Evidence is presented that short-chain phosphatidylcholines are superior to most detergents commonly used by biochemists. This is true not only regarding its excellent dispersing power on both phospholipid bilayers (Gabriel & Roberts, 1986) and biological membranes but also as to its capacity to preserve the native protein structure and hence enzymatic activity in the solubilized state. Due to its special properties DHPC lends itself very well not only to membrane solubilization but also to the purification of the solubilized membrane proteins and reconstitution of the proteins into simple lipid bilayers. Concerning the mechanism of membrane solubilization, evidence indicates that DHPC interacts primarily with the lipid bilayer of the membrane and not with the membrane proteins. DHPC solubilizes membranes by being distributed into the lipid bilayer and breaking it up. In the resulting small mixed micelles, the protein remains associated with its preferred intrinsic membrane lipids and is thus stabilized. The protein-intrinsic lipid complex is successfully shielded from unfavorable contacts with H2O by DHPC-intrinsic lipid interactions.