Lectin binding studies on murine peritoneal cells: physicochemical characterization of the binding of lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia to murine peritoneal cells

Purified 125I-labeled lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia (I-B4 isolectin) were used to analyze changes in the expression of carbohydrates on the surface of resident (PC) and thioglycollate-stimulated murine (C57B/6J) peritoneal exudate cells (PEC). The lectins from D. stramonium, E. europaea, and G. simplicifolia I-B4 bind specifically to PEC with relatively high affinity (Kd = 5.65 +/- 1.08 X 10(-7) M, 1.08 +/- 0.12 X 10(-8) M, and 1.33 +/- 0.15 X 10(-7) M, respectively). Assuming a single lectin molecule binds to each cell surface saccharide, the number of receptor sites per cell ranged for different cell samples from 22.3 to 50.0 X 10(6), from 3.8 to 4.8 X 10(6), and from 2.0 to 16.8 X 10(6) for D. stramonium, E. europaea, and G. simplicifolia I-B4 lectins, respectively. There were approximately 3- to 7-fold, 16- to 20-fold, and 2- to 20-fold increases in binding capacity for D. stramonium, E. europaea and G. simplicifolia I-B4, respectively, compared to the binding to resident, peritoneal cells. Scatchard plots of the binding of all three lectins to PEC were linear, suggesting that the receptor sites for these lectins are homogeneous and noninteracting. The binding capacity of these lectins to PEC was unchanged after trypsin digestion of cells. The expression of carbohydrates on the surface of PEC was also monitored by an agglutination assay. PEC were agglutinated by all three lectins whereas PC either were not agglutinated or were agglutinated only at high lectin concentrations. On the basis of our knowledge of the carbohydrate binding specificity of the D. stramonium and G. simplicifolia I-B4 lectins, we postulate that, parallel with thioglycolate stimulation, there is an increase in the number of N-acetyllactosamine residues and terminal alpha-D-galactosyl end groups. The blood group B, and H type 1 determinants--DGa1 alpha 1,3[LFuc alpha 1,2]DGa1 beta 1,3(or 4)DGlcNAc and LFuc alpha 1,2DGa1 beta 1,3DG1cNAc, respectively, as well as DGa1 alpha 1,3DGa1 beta 1,3(or 4)DGlcNAc--may be considered to be possible receptors for the E. europaea lectin. These glycoconjugates, present on the surface of peritoneal exudate cells, provide new chemical markers for studying the differentiation of resident peritoneal cells.     

Hydrogen peroxide formation and stoichiometry of hydroxylation reactions catalyzed by highly purified liver microsomal cytochrome P-450.

The stoichiometry of hydroxylation reactions catalyzed by cytochrome P-450 was studied in a reconstituted enzyme system containing the highly purified cytochrome from phenobarbital-induced rabbit liver microsomes. Hydrogen peroxide was shown to be formed in the reconstituted system in the presence of NADPH and oxygen; the amount of peroxide produced varied with the substrated added. NADPH oxidation, oxygen consumption, and total product formation (sum of hydroxylated compound and hydrogen peroxide) were shown to be equimolar when cyclohexane, benzphetamine, or dimethylaniline served as the substrate. The stoichiometry observed represents the sum of two activities associated with cytochrome P-450. These are (1) hydroxylase activity: NADPH + H⁺ + O₂ + RH → NADP⁺ + H₂O + ROH; and (2) oxidase activity: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂. Benzylamphetamine (desmethylbenzphetamine) acts as a pseudosubstrate in that it stimulates peroxide formation to the same extent as the parent compound (benzphetamine), but does not undergo hydroxylation. Accordingly, when benzylamphetamine alone is added in control experiments to correct for the NADPH and O₂ consumption not associated with benzphetamine hydroxylation, the expected 1:1:1 stoichiometry for NADPH oxidation, O₂ consumption, and formaldehyde formation in the hydroxylation reaction is observed.     

Studies on hydroperoxide-dependent substrate hydroxylation by purified liver microsomal cytochrome P-450.

Highly purified liver microsomal cytochrome P-450 catalyzes the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen. The addition of phosphatidylcholine is necessary for maximal activity. The absence of flavoproteins and cytochrome b₅ from the cytochrome P-450 preparations rules out the involvement of other known microsomal electron carriers. The ferrous form of cytochrome P-450 is not involved in peroxide-dependent hydroxylation reactions, as indicated by the lack of inhibition by carbon monoxide. With cumene hydroperoxide present, a variety of substrates is attacked, including N-methylaniline, N,N-dimethylaniline, cyclohexane, benzphetamine, and aminopyrine. With benzphetamine as the substrate, cumene hydroperoxide may be replaced by other peroxides, including hydrogen peroxide, or by peracids or sodium chlorite. A study of the stoichiometry indicated that equimolar amounts of N-methylaniline, formaldehyde, and cumyl alcohol (α,α-dimethylbenzyl alcohol) are formed in the reaction of N,N-dimethylaniline with cumene hydroperoxide. Since H₂¹⁸O is incorporated only slightly into cyclohexanol in the reaction of cyclohexane with cumene hydroperoxide, it appears that the oxygen atom in cyclohexanol is derived primarily from the peroxide. The data obtained are in accord with a peroxidase-like mechanism for the action of cytochrome P-450.     

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.     

Studies on Sudden Fatalities Among Piglets Following Parenteral Iron Therapy

An investigation was carried out in order to clarify whether there is a correlation between the latent iron-binding capacity, UIBG, in the serum of suckling piglets and sudden fatalities occurring among these animals when they are treated with 250 mg trivalent iron in the form of a complex also containing dextrin, sorbitol, citric acid and lactic acid. In all, 97 animals from 9 litters were used. By administering 100 mg oral divalent iron to 22 animals, the iron-binding capacity was saturated or appreciably reduced 3 hrs. after the oral treatment. After this time, the animals were treated with parenteral iron. Seventeen other animals were treated with 100 mg divalent iron and immediately afterwards with parenteral iron. Three hrs. later, the iron-binding capacity of the animals was exceeded. In 32 of the control animals, UIBG was high before the parenteral treatment. No fatalities were observed among the animals treated with parenteral iron. Twenty-three of the animals had a high iron-binding capacity in spite of having diarrhoea. On parenteral treatment of these animals with the iron complex, no fatalities were observed which could be ascribed to the treatment. The mechanism for the sudden fatalities among suckling piglets after parenteral administration of iron is discussed.     

A specific radioimmunoassay for androstenedione with reduced bridge-binding

Antibody used in a steroid radioimmunoassay raised against a steroid hapten-carrier protein conjugate may recognize both the hapten and the chemical bridge to the protein. Use of the same bridge in the radio-isotopic label may lead to higher affinity binding to the label than to the native steroid. Inhibition curves under these conditions are shallow and generally not acceptable for radioimmunoassay procedures. We have developed a radioimmunoassay for androstenedione that employs different bridges at the 11 beta position of the steroid for the protein conjugate and label. The resulting assay has greatly reduced bridge-binding, has an acceptable slope for the standard curve and is very specific as evidenced by low crossreactivies to other steroids.     

A chemical approach to solving bridging phenomena in steroid radioimmunoassays

Steroid radioimmunoassays (RIA) employ antibodies raised against a carrier protein-steroid conjugate. Individual antibodies may recognize the steroid, the protein or the chemical bridge used to Join them together. Use of the same bridge In the tracer results in higher affinity binding of the tracer than the native ligand which in turn results in a loss of sensitivity and precision. We have greatly reduced bridge-binding In a RIA for androstenedione. Conjugates and radioiodinated labels were prepared with either an ester op ether chemical bridge. By using an antibody and the corresponding label with the heterologous bridge very sensitive assays were obtained.