Contributions to the knowledge of African rotifers: Rotifers from Mauritania (W.-Africa)

The number of Rotifer taxa known from Mauritania, is raised from 8 to 90. 61% of these are cosmopolitan, 1 is temperate — boreal, 18% are thermophilic and 20% are tropicopolitan. Most of them are widely distributed in Africa, but some species have limited distributions. The presence of Keratella testudo and Wolga spinifera is remarkable and is probably related to the unusual climatic conditions in the SW Sahara. One phenotype or forma is new to science.     

The nifA gene of Rhizobium meliloti is oxygen regulated.

Experiments using plasmid-borne gene fusions and direct RNA measurements have revealed that expression from the nifA gene is induced in Rhizobium meliloti when the external oxygen concentration is reduced to microaerobic levels. Induction occurs in the absence of alfalfa and in the presence of fixed nitrogen and does not require ntrC. The production of functional nifA gene product (NifA) can be demonstrated by its ability to activate the nitrogenase promoter P1. Aerobic induction of nifA can also occur during nitrogen starvation at low pH, but in this case induction is dependent on ntrC and does not lead to P1 activation. The data indicate that reduced oxygen tension is potentially a major trigger for symbiotic activation of nitrogen fixation in Rhizobium species.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.     

Interaction of epidermal growth factor with micelles monitored by photochemically induced dynamic nuclear polarization-1H NMR spectroscopy

Photochemically induced dynamic nuclear polarization (CIDNP)-1H-NMR spectroscopy has been used to study the interaction of the protein hormone epidermal growth factor (EGF) with micelles of sodium dodecyl sulfate (SDS) and dodecylphosphorylcholine (DPC). Conventional 1H-NMR spectra show that most protein resonances remain unperturbed when micelles are added to solution, which argues that the overall protein conformation is maintained in the presence of SDS or DPC at the concentrations used. Photo-CIDNP enhancements of resonances assigned to aromatic side chains of residues at the COOH terminus and beta-sheet regions of murine EGF (i.e. Trp-49, Trp-50, and Tyr-37) are considerably reduced in the presence of micelles, while resonances of aromatic side chains of residues found elsewhere on the protein surface are mostly unaffected. This suggests that the primary interaction between murine EGF and the micelle occurs at the micelle-bulk solvent interface. The overall negatively charged surface of SDS micelles tends to induce a stronger interaction with the protein compared to the zwitterionic DPC micelles, probably due to electrostatic interactions. Cleavage of the COOH-terminal pentapeptide containing both tryptophan residues enhances the already present, but weak, interaction with Tyr-10 and attenuates it with Tyr-37. A similar interaction pattern is found with rat EGF suggesting that at least concerning these two species of EGF the interaction is somewhat specific and conserved. A simple mass-action model for protein-micelle interaction is also presented.     

Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

Role of the hinge protein in the electron transfer between cardiac cytochrome c1 and c. Equilibrium constants and kinetic probes

A role of the hinge protein is studied in the electron transfer reaction between cytochromes c1 and c, using highly purified "one-band" cytochrome c1 and "two-band" cytochrome c1. The results show that the hinge protein (Hp), which is essential for a stable ionic strength-sensitive c1-Hp-c complex, seems to play a certain role in electron transfer between cytochromes c1 and c; Keq for electron transfer reaction between cytochromes c1 and c in the presence of the hinge protein is found to be about 40% higher than that in the absence of the hinge protein at low ionic strength, but no difference exists at high ionic strength. We propose a hypothesis that the hinge protein may function as regulator for the electron transfer reaction between cytochromes c1 and c, and this may be at least one of the roles of the hinge protein in mitochondria.     

Heparan sulfate proteoglycans of human lung fibroblasts. Structural heterogeneity of the core proteins of the hydrophobic cell-associated forms

Heparan sulfate proteoglycans (HSPG) were solubilized from human lung fibroblast monolayers with detergent. Presumptive membrane-associated forms displaying hydrophobic properties were purified by gel filtration on Sepharose CL-4B, by ion-exchange chromatography on Mono Q and by incorporation in lipid vesicles. The HSPG preparations were 125I-iodinated and treated with heparitinase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five radiolabeled proteins with apparent molecular weights of 125,000, 90,000, 64,000, 48,000, and 35,000 were visualized by autoradiography. A sixth protein, identified in nonreduced 125I-HSPG preparations, appeared as a non-HS chain-bearing Mr 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of similar size. This multiplicity of core proteins did not seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins migrated independently during gel filtration before heparitinase digestion. Moreover, heparitinase digestion of 125I-HSPG purified by affinity chromatography on an immobilized monoclonal antibody yielded only the Mr 64,000 protein. Alternative depolymerizations of the HS chains by heparinase or HNO2 also yielded multiple protein bands. These results imply that heterogeneity of the core protein moiety may be a genuine property of the hydrophobic HSPG of human lung fibroblasts. The occurrence of multiple integral membrane HSPG forms may be relevant for the multiple functions that have been ascribed to cell-surface HSPG.