Establishing isostructural metal substitution in metalloproteins using 1H NMR, circular dichroism, and Fourier transform infrared spectroscopy.

Far-UV CD, 1H-NMR, and Fourier transform infrared (FTIR) spectroscopy are three of the most commonly used methods for the determination of protein secondary structure composition. These methods are compared and evaluated as a means of establishing isostructural metal substitution in metalloproteins, using the crystallographically defined rubredoxin from Desulfovibrio gigas and its well-characterized cadmium derivative as a model system. It is concluded that analysis of the FTIR spectrum of the protein amide I resonance represents the most facile and generally applicable method of determining whether the overall structure of a metalloprotein has been altered upon metal reconstitution. This technique requires relatively little biological material (ca. 300 micrograms total protein) and, unlike either CD or 1H-NMR spectroscopy, is unaffected by the presence of different metal ions, thus allowing the direct comparison of FTIR spectra before and after metal substitution.     

The interaction of gadolinium complexes with isolated rat hepatocytes

The lanthanide metal, gadolinium, is currently used in contrast agents for magnetic resonance imaging. We have performed a study of the interaction between isolated rat hepatocytes and ¹⁵³Gd complexed to diethylene-triamine pentaacetic acid (DTPA) or to DTPA-albumin conjugates. The study shows that isolated hepatocytes are able to take up both types of ¹⁵³Gd complexes. The ¹⁵³Gd-DTPA-albumin complexes are apparently taken up by pinocytosis, and possibly receptor-mediated endocytosis and/or adsorptive endocytosis, whereas the uptake mechanism of ¹⁵³Gd-DTPA is unknown. The ¹⁵³Gd-DTPA-albumin complexes, but not the ¹⁵³Gd-DTPA complex, are degraded by the cell. The degradation is inhibited by ammonium chloride. Gadolinium is slowly released back to the medium after loading of the cells with both complex types. In the experiments reported here no evidence of any adverse effects on the hepatocyte resulting from exposure to the ¹⁵³Gd-complexes were observed.     

Responses of the teleost Hoplias malabaricus to hypoxia

Synopsis Oxygen uptake (VO₂) during graded hypoxia, rate of hypoxia acclimation, breathing frequency (f_R), breath volume (V_{S, R}) and gill ventilation (V_G) were measured in Hoplias malabaricus. Normoxia and hypoxia acclimated fish had similar and constant VO₂ and V_G in a range of water PO₂ from 150 to 25 mmHg. Hypoxia acclimated fish showed significantly higher VO₂ in severe hypoxia (PO₂ <15 mmHg). Normoxia acclimated fish showed symptoms similar to hypoxic coma after 1 h of exposure to water PO₂ of 10 mmHg whereas the same symptoms were observed only at PO₂ of 5 mmHg for fish acclimated to hypoxia. Fish required 14 days to achieve full acclimation to hypoxia (PO₂ ≥25 mmHg). Lowering of water PO₂ from 150 to 25 mmHg resulted in normoxic fish showing a 3–2 fold increase in V_G. The increase was the result of an elevation in V_{S, R} rather than f_R. Among normoxia acclimated specimens, small fish showed a higher V_G per unit weight than the large ones in both normoxia (PO₂ =150 mmHg) and hypoxia (PO₂ = 15 mmHg). A decrease in the ventilatory requirement (V_G/VO₂) with increased body weight was recorded in hypoxia (PO₂ = 15 mmHg).     

Biomass yields of Chlorella from iron (Y x/Fe) in iron-limited batch cultures

The maximum biomass in iron-limited photosynthetic batch cultures of chlorella increased as the logarithm of the iron concentration. The growth yield from iron (Y _x/Fe) showed a marked inverse relation to the specific growth rate. The maximum biomass yield, g dry biomass/g iron consumed, was 7.5x10³ with specific growth rate 0.108 h^-1; the minimum was 0.79×10³ with specific growth rate 0.145 h^-1. The maximum specific growth rate in the exponential phase of Fe limited cultures varied as the initial Fe concentration. Fe-limited growth made the cells adhere to a glass surface.Abbreviation O.D. optical density     

Stimulation of growth and glucose catabolite enzymes by succinate in some thermophilic fungi

Thermophilic Humicola lanuginosa, Penicillium duponti, Sporotrichum thermophile and Mucor pusillus required succinate in addition to glucose for optimal growth. The requirement for succinate was concentration-dependent and the concentration needed for one half of the maximal growth was 6.14 mM. In the presence of succinate, glucose utilization from the medium was markedly increased and this was associated with increased levels of the enzymes of the glycolytic and Krebs cycle pathways. Addition of succinate to cultures growing in glucose at any stage of growth stimulated the growth with the resulting rate of growth remaining high if the addition was made within 3 days of inoculation. Cycloheximide (71.4 M) prevented the succinate-mediated derepression of the enzymes suggesting that succinate may remove the catabolite repression in the presence of glucose.A preliminary part of this work was presented at the 17th annual meeting of the Association of Microbiologists of India at Manipal (India) held from Dec. 13 to 15, 1976     

Nodulation and growth ofLablab purpureus (Dolichos lablab) in relation to rhizobium strain,liming and phosphorus

Summary Greenhouse experiments were done with two purposes: (1) to identify strains of rhizobia effective and acid-tolerant in symbiosis withLablab purpureus, and (2) to determine whether soil acidity or the symbiotic condition increased the phosphate requirement for growth.Five rhizobial strains were tested in one neutral soil, two acid soils, and the two acid soils limed to pH 6.6. In the neutral and limed soils, three of the strains were effective (CB1024, CB756, TAL169), but only two strains (CB756, TAL169) remained effective in acid soil.Strain CB756 and plus-N treatments were further compared in a factorial trial involving combinations of five levels of P with lime, no lime and CaCl₂ treatments, applied to an acid soil. Some of the treatments were also applied to plants inoculated with CB1024. Between the N-fertilized and CB756 treatments there was no clear difference in growth response to applied P, and the critical internal concentration of P for 95% of maximal growth was the same (0.22% shoot dry weight). Increasing P beyond levels needed for maximal growth increased nodulation and N concentration in plants inoculated with CB756. It lowered N concentration in N-fertilized plants. There was evidence suggesting that the P requirement of symbiotic plants increased if the soil was acid, or if CB756 were replaced by CB1024 as microsymbiont; but the critical statistical interactions were not significant.     

The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus

The initiation of sporangiophores of Phycomyces was analyzed under oxygen-limiting conditions. Mutants lacking -carotene have a higher oxygen threshold than the wild type depending on the residual amount of -carotene. The supersensitivity to low oxygen tension is specific for sporangiophore initiation and can be suppressed by addition of either retinal, retinol or retinol acetate to the medium. It is suggested that retinol is a natural regulator of differentiation in Phycomyces.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Effects of lead and cadmium on chemical composition and total water content of the pupal parasitoid, Pimpla turionellae

The parasitoid Pimpla turionellae L. (Hymenoptera, Ichneumonidae) was fed on Cd, Pb and Cd+Pb-contaminated food (33g Cd, 82g Pb and 33g Cd+82g Pb per gram food fresh weight, respectively). Significant decrease in the total lipid and protein content was found along with an increase in the water content particularly in Cd-contaminated parasitoids.     

Why the evolution of resistance to anthropogenic toxins normally involves major gene changes: the limits to natural selection

Standard population genetic theory suggests that adaptation should normally be achieved by the spread of many genes each of small effect (polygenes), and that adaptation by major genes should be unusual. Such models depend on consideration of the rates of acquisition of adaptation. In practice, adaptation to pollutants and anthropogenic toxins has most frequently been achieved by the spread of major genes. A simple model is developed to explain this discrepancy, in which the determining factor is not the rate of spread, but the maximum response achievable under the two contrasting models of polygenic or major gene inheritance. In the short term, for a given mean and genetic variance, characters in which the additive genetic variance is produced by the segregation of many genes of small effect at intermediate gene frequencies are unable to produce as large a response to directional selection as characters in which the variance is caused by genes of large effect at low frequency. If the target for selection is a long way from the mean prior to selection (as it may well be for adaptation to novel anthropogenic stresses) then adaptation can only be achieved by species possessing major genes. The model is discussed with reference to the example of heavy metal tolerance in plants.