Isolation and properties of crystalline ferredoxin from lactuca sativa

Lettuce ferredoxin has been purified to homogeneity, with a yield of 18 mg/kg of denerved leaves. It crystallizes in magnificent needles, often clustered in broom-like sheaves. The absorption spectrum showed maxima at 460, 422, 330 and 274 nm,with a ratio A₄₂₂/A₂₇₄, of 0.46. The mM absorption coefficient was 9.74 at 422 nm, and 21.62 at 274 nm. This ferredoxin showed a pI = 4.7 and an E′₀ = ?425 mV (at pH = 7.7). MWs of 12 400, 11480 and 13000 were obtained by sucrose gradient centrifugation, and on the basis of the amino acid composition and the iron content, respectively, with an average of 12 300. The amino acid analysis showed the existence of one methionine residue per mole, with 105 amino acid residues. There are two iron atoms and two labile sulfide groups per mole; 4 half-cystine residues were found by performic acid oxidation, and 5 cysteine groups when determined by titration with pHMB. The native protein is not fixed on thiol-Sepharose 4B, but it is quantitatively retained after incubation with 8 M urea. Lettuce ferredoxin showed a 62, 58 and 78% effectiveness with the spinach ferredoxin-NADP reductase, nitrite reductase and fructose-1,6-diphosphatase (FDPase), respectively, when compared with the spinach ferredoxin. This different behaviour of both ferredoxins is joined to genetic-structural relationships, and suggests that the role of ferredoxin in FDPase activation is more sophisticated than that of a mere nonspecific reductant.     

Isolation and immunological characterization of a glucose-regulated fibroblast cell surface glycoprotein and its nonglycosylated precursor.

We have isolated and immunochemically characterized a major membrane glycoprotein of mouse 3T3 cells. This GRP (glucose/glycosylation-regulated protein) is labeled by lactoperoxidase-mediated iodination and by ¹⁴C-glucosamine, binds concanavalin A and has an apparent molecular weight in SDS-polyacrylamide gels of 92,000 daltons (or 97,000 daltons in a discontinuous gel system). Glycosylated GRP was isolated from plasma membranes using Triton X-100 extraction, affinity chromatography on concanavalin A-Sepharose and preparative SDS gel electrophoresis.Antibody against this glycosylated GRP stains the external surfaces of mouse cells and induces patches and caps. Immunofluorescence and immunoprecipitation studies indicate that this glycoprotein can exist in the membrane in two molecular forms, either as a glycosylated or as a nonglycosylated protein. The nonglycosylated form is induced under conditions of limited glycosylation or glucose deprivation. This nonglycosylated GRP remains accessible to antibodies on the exterior of cells, but becomes inaccessible to lactoperoxidase.The immunoprecipitation of the 92K GRP with its specific antibody is always associated with the precipitation of a small fraction of the other major GRP of molecular weight 75,000 daltons. We suggest that both GRP (92K and 75K) may function in close association in the membrane.     

Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen.

Fourteen different broth media were autoclaved under anaerobic conditions and then exposed to atmospheric oxygen. The hydrogen peroxide and superoxide radical formation as well as the bactericidal effect of the media were studied. The rate of killing of Peptostreptococcus anaerobius VPI 4330-1 was high in media that rapidly autoxidized and accumulated hydrogen peroxide. In actinomyces broth (BBL), 50% of the cells were killed within 2 min, and in Brewer thioglycolate medium (Difco), 50% were killed within 11 min, whereas more than 50% of the cells survived for more than 2 h in Clausen medium (Oxoid), fluid thioglycolate medium (BBL), and thioglycolate medium without dextrose or indicator (Difco). Only media that contained phosphate and glucose had a tendency to accumulate hydrogen peroxide. A solution of phosphate and glucose autoxidized when it had been heated to 120 degrees C for at least 5 min and when the pH of the solution was higher than 6.5. Transitional metal ions catalyzed the autoxidation, but they were not necessary for the reaction to occur. Of the other substances heated in phosphate buffer, only alpha-hydroxycarbonyl compounds autoxidized with accumulation of hydrogen peroxide. Superoxide dismutase decreased the autoxidation rate of most of the broth media. This indicated that superoxide radicals were generated in these media.     

Excretion of citric and isocitric acids by the yeast Saccharomycopsis lipolytica

Summary We investigated the excretion of citric and isocitric acids in a strain of Saccharomycopsis lipolytica grown on either n-paraffins, glucose, or glycerol. These acids were excreted in the ratio of 67:33 on n-paraffins and roughly 92:8 on either glucose or glycerol. However, with all the carbon sources used, the relative amount of isocitric acid in the intracellular pool remained below 10%. The assimilation of citric and isocitric acids was prevented when glucose or glycerol were the carbon sources, but not when n-paraffins were used. Citric acid stopped isocitric acid assimilation. These phenomena of selective assimilation and/or uptake might explain the variations observed in the ratio of citric to isocitric acids excreted on different carbon sources.     

Effect of prostaglandins E1 and F2 alpha on neuromuscular transmission in the frog sartorius muscle.

End plate potentials (e.p.p.s.) and miniature end plate potentials (m.e.p.p.s.) were recorded intracellularly at the neuromuscular junction of the frog sartorius muscle. Addition of as little as 8.5 x10(-8)M PGE1 reduced the mean m.e.p.p. frequency. The mean amplitude of m.e.p.p.s was not changed, the mean amplitude of the e.p.p.s and the quantum content of the transmitter released by a nerve impulse was slightly reduced. A decrease in mean m.e.p.p. frequency was also seen in response to the administration of 8.5 x 10(-8)M PG2 alpha. The mean amplitude of e.p.p.s and m.e.p.p.s and the quantum content remained unchanged. The possible presynaptic mode of action of PGs in the preparation of discussed.     

Inducible α-glucosidase of Mucor rouxii: Effects of dimorphism on the development of the wall-bound activity

Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed.