Presence Of Reductant Research Articles

The subunit structure of the high affinity insulin receptor. Evidence for a disulfide-linked receptor complex in fat cell and liver plasma membranes.

125I-Insulin equilibrated with high affinity fat cell and liver plasma membrane receptors was cross-linked to the membrane by addition of disuccinimidyl suberate. Autoradiographic analysis of the 125I-insulin-linked membranes subsequent to dodecyl sulfate electrophoresis in the absence of reductant revealed the presence of one labeled species which migrated with an apparent molecular weight of 300,000. Electrophoresis of these membranes in the presence of dithiothreitol resulted in the appearance of one major labeled band of about Mr = 125,000 concomitant with the loss of label in the Mr = 300,000 region. Another radioactive species which migrated in the Mr = 225,000 region on the reduced gels contained a much smaller amount of label. The degree to which the Mr = 125,000 membrane component was cross-linked to 125I-insulin in these experiments paralleled the extent of occupancy of high affinity membrane receptors by the 125I-insulin. 125I-Insulin-linked plasma membranes derived from adipocytes alkylated with N-ethylmaleimide prior to homogenization to prevent spontaneous sulfhydryl oxidation also exhibited the Mr = 300,000 and 125,000 labeled bands upon electrophoresis in the absence and presence of reductant, respectively. These same radioactive species were observed when 125I-insulin was covalently corss-linked to intact adipocytes. These data indicate that the labeled monomer with an apparent molecular weight of 125,00 represents a high affinity insulin receptor subunit which exists in the native adipocyte plasma membrane in a disulfide-linked complex. The amount of cross-linked label present in the reduced form of the receptor (Mr = 125,000) was only 20 to 30% of that present in the oxidized species (Mr = 300,000). Since the 125I-insulin used in these studies is labeled predominantly on the A chain, this large loss of label upon reduction indicates that it is predominantly the B chain of insulin that is covalently linked to the receptor protein by disuccinimidyl suberate. 125I-Proinsulin which lacks an A chain NH2 terminus could also be cross-linked to the receptor, indicating that the A1 glycine is not critical to the cross-linking reaction. 125I-Insulins modified either at lysine B29 with an acetyl group or at phenylalanine B1 with a phenylthiocarbamoyl group were both readily cross-linked to the receptor with roughly equal efficiency. It is concluded that the B1 terminal amino and B29 lysine amino groups are both accessible for cross-linking of insulin to the receptor by disuccinimidyl suberate.

Mechanism of iron uptake by plants

Abstract. Green plants require a continuous supply of Fe as they grow, because Fe does not not move from the older to the newer leaves. Soils do not lack Fe per se, but it may not be available to plants grown in alkaline soils. Plants are classed ‘Fe‐efficient’ if they respond to Fe‐deficiency stress by inducing biochemical reactions that make Fe available in a useful form, and ‘Fe‐inefficienT’ if they do not. Iron uptake induced in response to Fe stress involves release of hydrogen ions and reductants by the root. The lowered pH and presence of reductant at the root zone, along with reduction of Fe3+ to Fe2+ at the root surface, enables Fe2+ to be taken up primarily through the young lateral roots. Ferrous iron is present throughout the protozylem and may or may not have entered the root by a carrier. The root‐absorbed Fe2+ is oxidized to Fe3+ at the junction of the protoxylem and the metaxylem, chelated by citrate, and then transported in the metaxylem to the plant top. In the plant, the chemical reactions injuced by Fe‐deficiency stress may affect nitrate reductase activity, use of Fe from Fe3+ phosphate and chelating agents, and tolerance to heavy metals. An efficient mechanism for Fe uptake in roots appears to be important for the efficient use of Fe in plant tops.

Thiol-dependent subunit interactions of a major fat cell plasma membrane glycoprotein.

A major glycoprotein fraction of the isolated adipocyte plasma membrane migrated in the 200,000-dalton region of dodecyl sulfate polyacrylamide gels following solubilization in 2% dodecyl sulfate/4M urea at room temperature in the absence of reductant. Limited heat treatment allowed resolution of the glycoprotein into two distinct bands in the same high-molecular-weight region plus a new 94,000-dalton glycoprotein band. Prolonged incubation of solubilized plasma membranes at 100 degrees C for 15-30 min or incubation with reductant resulted in complete conversion of the high-molecular-weight bands to the 94,000-dalton region, indicating dissociation of dimers to the monomeric form. When the dimer bands on column gels were electrophoresed in the second dimension on slab gels in the presence of reductant, no low-molecular-weight bands were observed other than that in the 94,000-dalton region. The effects of limited heat treatment to permit resolution of the two dimers and the extended treatment to convert the dimers to the monomeric form were markedly inhibited by alkylation of the solubilized membrane protein with N-ethylmaleimide or oxidation with H2O2 or diamide. However, these latter treatments did not prevent complete dissociation of dimers due to addition of reductants. These results suggest that two glycoprotein fractions may exist as dimers in the native fat cell plasma membrane. The data are consistent with a model in which the glycoprotein subunits are linked by hydrophobic bonds that are sensitive to reduction of intramolecular disulfides but are stabilized by alkylation or oxidation of the glycoprotein sulfhydryls.

Ligand-induced spectral changes in cytochrome c oxidase and their possible significance

1. The spectral shifts induced on the binding of H2S to ferric cytochrome aa3 are similar to those induced by cyanide, reflecting a possible high- to low-spin state change in the a3 haem. Opposite shifts are seen with either formate or low azide concentrations, while high azide concentrations reverse the change induced at lower concentrations. The unusually high Soret band in the half-reduced sulphide-inhibited species (a2+a33+H2S) results from the superposition of cytochrome a2+ and cytochrome a33+H2S peaks. 2. The difference spectra in the visible region for cytochrome a2+ minus cytochrome a3+ obtained with four inhibitors (cytochrome a2+ a3+I minus minus a3+a33+I)are similar, except that azide and sulphide induce blue shifts of the alpha-peak. The trough in the Soret region for the azide complex is much deeper than that for the other complexes, suggesting changes in the cytochrome a33+HN3 centre on reduction of cytochrome a. 3. The "oxygenated" and "high-energy" forms of cytochrome aa3 both involve spectral changes at the a3 haem similar to the changes induced by cyanide and sulphide. The spectrum of partially reduced cytochrome aa3 in the presence of reductant and oxygen indicates the steady-state occurrence of appreciable levels of low-spin (oxygenated) cytochrome aa3. These may be important for energy conservation during the action of cytochrome aa3 in the intact mitochondrial membrane.

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide.

1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.

The Effect of Reductant in Inorganic Phosphate Release from Adenosine 5'-Triphosphate by Purified Nitrogenase of Clostridium pasteurianum

The two components of nitrogenase of Clostridium pasteurianum, azoferredoxin and molybdoferredoxin, together with an electron donor, and energy source, and Mg++ are required for ATP-dependent H2 evolution and reductant-dependent ATP utilization. ATP is also utilized by this N2-fixing system without H2 evolution and without the need for an electron donor. For this activity there is still an absolute requirement for both azoferredoxin and molybdoferredoxin. The ratio of ATP utilized in the absence of reductant to that in the presence of reductant was approximately 1:3 at pH 6.6, whereas at pH 5.2 there was little difference between the two ATP-consuming activities. The ratio of ATP consumed to H2 evolved by the N2-fixing system at pH values between 6.0 and 7.6 varied between 3 and 8. At pH 6.6 a minimum ratio of 3 was obtained. No H2 was evolved in the absence of an electron donor even though ATP was consumed. When the amount of ATP used for each H2 evolved at all pH conditions was corrected for the amount of ATP used in the absence of the reductant, a constant ratio of 2 ATP per H2 evolved was obtained. The visible spectrum of molybdoferredoxin between 300 to 400 nm also changes with pH, suggesting that a certain form of the chromophore of molybdoferredoxin may be required for the reductant-dependent utilization of ATP.