Chaotropic resolution of high molecular weight (type I) NADH dehydrogenase, and reassociation of flavin-rich (type II) and flavin-poor subunits.

1. Type-I NADH dehydrogenase (Complex I) was solubilized and dissociated into subunits by NaClO4. NADH slows the dissociation. On subsequent stepwise addition of (NH4)2SO4 the dissociation is partly reversed, as is to be expected from the opposing effects of ClO-4 and SO-24, which are on the salting-in and salting-out sides, respectively, of the lyotropic series. 2. In consequence, the aggregates of subunits that are separated by (NH4)2-SO4 fractionation consist of randomly associated subunits as well as fragments of Type I enzyme. The fraction precipitating at 27% satd. (NH4)2SO4 is flavin-poor, that remaining soluble at 55% satd. (NH4)2SO4 flavin-rich and those separating between 27 and 55% satd. (NH4)2SO4 intermediate in composition. 3. The fraction remaining soluble at 55% satd. (NH4)2SO4 contains the purified low-molecular-weight iron-sulphur flavoprotein (Type-II dehydrogenase). It is a dimer consisting of one molecule of FMN, one 28-kilodalton and one 56-kilodalton subunit per protomer. Work of others indicates that it contains 4 Fe and 4 acid-labile S atoms per molecule of FMN. Sometimes the fraction remaining soluble at 55% satd. (NH4)2SO4 contained an additional small subunit (12 kilodaltons) and four additional Fe and acid labile S atoms per protomer. The sedimentation coefficients (s020,w) of the two preparations were 5.3 and 6.6 S, respectively, with calculated frictional ratios of 1.5 and 1.24, respectively. 4. The intermediate fractions are mixtures of the various subunits present in Complex I. Specifically a fraction separating at 55% satd. (NH4)2SO4 was found to be a mixture of two fragments, the pure iron-sulphur flavoprotein and a 26-S fragment that contained per protomer four subunits of 12 kilodaltons, one each of 28, 32, 56 and 77 kilodaltons, one molecule of FMN and 20 Fe and acid-labile S atoms. It was probably tetrameric or even larger. 5. The oxidoreductase activity of the intermediate fractions is dependent on the protein concentration, the activity with ferricyanide increasing and that with ferricytochrome c decreasing with increasing protein concentration. This is interpreted as an increased association of subunits present in the intermediate fractions. Similar results are obtained when flavin-rich and flavin-poor fractions are mixed. The association is cooperative. NADH favours the association of the subunits. 6. Association of the subunits is accompanied by a 10-fold increase in k2 (rate constant for intramolecular electron flow), a 10-fold decrease of the accessibility of ferricyanide to the reduced enzyme and a 10(4)-fold decrease of the accessibility of ferricytochrome c. The Ks (NADH) is also decreased. Although the changes are in the direction to be expected from a conversion of Type II enzyme to Type I, the value of k2 is still much less than in the latter enzyme.     

Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease.

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles

The rate of transbilayer movement of dioleoylphosphatidylcholine in sonicated lipid vesicles is enhanced by at least two orders of magnitude upon incorporation of glycophorin in the bilayer.     

Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.     

Determination of the number of cross-links in a protein gel from its mechanical and swelling properties

The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.     

T1 and T2 lymphocytes in primary and secondary delayed type hypersensitivity of mice. I. Contribution in the response to sheep red blood cells and to allogeneic spleen cells.

Peripheral T lymphocytes can be subdivided into two populations (T₁ and T₂ cells) based upon the short life span of T₁ cells after adult thymectomy (ATx) and sensitivity of T₂ cells to treatment with anti-thymocyte serum (ATS) in vivo. The contribution of the T₁ and T₂ cells to primary and secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) and to primary DTH to allogeneic cells was studied in mice. T₂ cells were found to account for the development of the state of primary DTH responsiveness after intravenous immunization with SRBC and after subcutaneous immunization with allogeneic cells. No clear cut evidence was found that in the presence of T₂ cells DTH related T effector cells were generated from T₁ cells. In mice selectively depleted for T₁ cells by ATx, the remaining T₂ cells were capable to generate SRBC-specific T memory cells, but not in numbers as large as in non-thymectomized mice. On the other hand, T₁ cells in mice depleted for T₂ cells by ATS treatment, could give rise to normal numbers of SRBC-specific T memory cells. Apparently T₁ cells can compensate for the absence of T₂ cells during generation of T memory cells, but T₂ cells cannot do so for the loss of T₁ cells. From the time curve showing the ATx-induced decline of the population of SRBC-specific T₂ cells, involved in primary DTH responsiveness, the half life was calculated to be 6 to 7 months.     

Accumulation of carnitine esters of beta-oxidation intermediates during palmitate oxidation by rat-liver mitochondria

Rat-liver mitochondria were incubated with [14C]palmitate in the presence of L-malate, fluorocitrate, and L-carnitine. The specific activities of acetyl groups incorporated into citrate, ketone bodies and acetyl-L-carnitine were measured. During state-4 oxidation of [1--14C]palmitate the specific activity of the acetyl-CoA pool was 1.3-times higher than that of the average acetyl group of palmitate, indicating an incomplete breakdown of the palmitate molecule. Accumulation of carnitine esters was observed in this condition. The acyl moieties of carnitine esters formed during the state-4 oxidation of [U-14C]palmitate or [16(-14)C]palmitate were analysed by radioactive gas-chromatography. Substantial amounts of beta-oxidation intermediates were found. The accumulation of carnitine esters of C6-C14 intermediates can quantitatively explain the high specific activity of the acetyl-CoA pool during the state-4 oxidation of [1(-14)C] palmitate. The localization and control of beta-oxidation are discussed.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.