Interactions of phosphorylethanolamine analogs with phosphorylethanolamine-citidylyltransferase]

Kinetic studies of ethanolaminephosphate-cytidylyltransferase (E.C. 2.7.7.14) from rat liver have been carried out in presence of structural analogues of ethanolaminephosphate : these compounds acted as inhibitors of the enzyme: - 2-aminoethylphosphonate behaved as a substrate and a competitive inhibitor to phosphorylethanolamine: the Km value of 2-aminoethylphosphonate was nearly the same as its Ki value, at pH = 5,5 (30 X 10(-3) M and 24 x 10(-3) M, respectively). - 3-aminopropylphosphonate was also a competitive inhibitor. It appeared to be the best inhibitor at pH optimum (pH = 7,7). - 1-aminoethylphosphonate behaved as a noncompetitive inhibitor. However, cytidylyltransferase was relatively specific, inhibitions being always weak. Inhibitory power of phosphonates was stimulated by Mg++.     

Interactions between carbamyl phosphate synthase-I-mitochondrial aspartate aminotransferase and palmitoyl-CoA

Carbamyl phosphate synthase-I and glutamate dehydrogenase both form a complex with mitochondrial aspartate aminotransferase. Instead of these two enzymes competing for the aminotransferase, carbamyl phosphate synthase-I enhances glutamate dehydrogenase-aminotransferase interaction. This suggests that a complex can be formed between all three enzymes. Since this complex is stable in the presence of substrates and modifiers of the three enzymes, it could conceivably convert NH₄⁺ produced from aspartate into carbamyl phosphate. Furthermore, since carbamyl phosphate synthase-I is the predominant protein in liver mitochondria, it could play a major role in placing the aminotransferase and glutamate dehydrogenase in close proximity. Malate removes glutamate dehydrogenase from the tri-enzyme complex and thus could play a role in determining whether glutamate dehydrogenase interacts with carbamyl phosphate synthase-I or is available to participate in reactions with the Krebs cycle. Palmitoyl-CoA has a high affinity for both carbamyl phosphate synthase-I and glutamate dehydrogenase. ATP and malate which, respectively, decrease and enhance binding of palmitoyl-CoA to glutamate dehydrogenase, respectively decrease and enhance the ability of this enzyme to compete with carbamyl phosphate synthase-I for palmitoyl-CoA. Since carbamyl phosphate synthase-I is present in high levels in liver mitochondria and has a high affinity for palmitoyl-CoA, it could play a major role as a reservoir for palmitoyl-CoA.     

Interactions between polynucleotides and platinum (II) complexes

Reaction of either $\text{cis}$ or $\text{trans}$ Pt(NH₃)₂Cl₂ with poly(A) in dilute aqueous solution leads to quantitative precipitation of the polymer at Pt/nucleotide ratios above 0.5. It is proposed that at ratios less than this, intramolecular binding of one Pt to two bases is favored; at higher ratios, intermolecular cross-linking becomes important and precipitation results. The absence of isomer selectivity in precipitation implies that the biological specificity of the $\text{cis}$ form results from a process other than cross-linking of polynucleotide strands. Other observations suggest that the coordinated ammonia of nucleotide-platinum(II) ammine complexes may be unusually labile.     

Toad egg-jelly as a source of divalent cations essential for fertilization

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boer's solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.     

Cytochrome c interaction with membranes. II. Comparative study of the interaction of c cytochromes with the mitochondrial membrane

A comparative study of the interaction of various cytochromes c with phospholipid vesicles and with mitochondrial membranes was undertaken. Both mammalian and yeast types of cytochrome c bind preferentially in the oxidized form as evidenced by the midpoint redox potential (E_m 7.0) becoming more negative upon binding. Cytochrome c which is reincorporated into cytochrome c-depleted mitochondria is kinetically comparable with the native cytochrome c component; rate of cytochrome b oxidation is maximally restored at ratios of c₁:c:a of 1:1:1. Comparison between the electron paramagnetic spectrum of cytochrome c labeled at methionine 65 or cysteine 103 reveals that upon binding to the mitochondrial membrane, the former is immobilized and not the latter. This result suggests that cytochrome c binds to the membrane at the side at which methionine 65 is located.     

Fractionation of plant chromatin with immobilized RNA-polymerase.

A new heptasaccharide, lacto-N-fucoheptaose has been isolated from human milk. It contains D(+)-galactose, D(+)-glucose, L(?)-fucose and N-acetyl-D-(+)-glucosamine in a 3 : 1 : 1 : 2 ratio. The glucose residue is at the reducing end of the oligosaccharide. Data obtained by partial acid hydrolysis, permethylation and enzymic hydrolysis establish the structure of lacto-N-fucoheptaose as follows:

     

Classification of interferons with antibody to immune interferon

Mouse immune Interferon, induced by the T-cell mitogen staphylococcal enterotoxin A (SEA), was partially purified and used to immunize rabbits. The resulting antiserum neutralized all immune interferon preparations tested, including interferon induced in vitro by SEA, concanavalin A, phytohemagglutinin P, and pokeweed mitogen, and in mixed lymphocyte cultures. Interferon produced in vivo with specific antigen was also neutralized. The antiserum was equally potent against all these interferon preparations. The serum did not neutralize any virus-type interferon preparation tested, but immune interferon induced by SEA in athymic nude mouse spleen cells was neutralized. The neutralizing activity was precipitable by 33% ammonium sulfate, and was not removed by absorption of the serum with mouse cells. The data suggest that immune interferons produced under diverse conditions are antigenically the same or closely related.     

Reaction of cyanide with glutathione peroxidase

An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 mM KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from [¹⁴C]iodoacetate in equimolar ratio to enzyme selenium. These observations suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and S-cyanoglutathione; spontaneous oxidation of the selenol to a labile oxidized form of GSH peroxidase leads to irreversible inactivation.     

The interaction of cells persistently infected with canine distemper virus with antiviral antibody and complement

Cells persistently infected with canine distemper virus can be lysed by antibody and complement. This reaction is dependent upon the alternative complement pathway. In the absence of antiviral antibody, the virus-infected cells will activate the alternative complement pathway, although this will not produce cell lysis.     

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.     

Marrow-dependent cell function in early stages of infection with Listeria monocytogenes.

Mice were infected with either Listeria monocytogenes (LM) or Yersinia pestis EV 76 stain (YP), which are facultative intracellular and extracellular organisms, respectively. Bacterial growth in spleen was determined at various intervals following challenge, focusing particularly on the critical period prior to the emergence of specific immunity. Natural resistance to LM during the first 2 days was diminished by treatment of adult mice with ⁸⁰Sr or silica particles, but not by treatment with lethal total-body irradiation, cortisol, or cyclophosphamide (CY). Early stages of resistance to YP were unaffected by ⁸⁰Sr, but were reduced by lethal total-body irradiation, silica particles, cortisol, and (CY). Infant mice manifested no resistance comparable to that of adults against LM prior to 19 days of age, whereas resistance against YP was attained by 14 days of age. The data are consistent with the hypothesis that marrow-dependent (M) cells function in host defense against early stages of infection with LM but not with YP.     

Characterization of a monoclonal antibody reactive with rabbit T lymphocytes and neutrophils

An IgG1 monoclonal antibody, termed ACM-1, has been shown to react with rabbit T cells, but not Ig+ cells or macrophages. This antibody appears to recognize the same epitope as the previously described 9AE10 antibody and, together with 9AE10, has been used to obtain highly pure and fully functional T- and B-cell populations. However, the relevant epitope does not appear to be homologous to rodent Thy-1 since quantitative absorptions failed to show reactivity with rabbit brain. Furthermore, attempts to obtain in vivo T-cell depletion resulted in larger decreases in white blood cells than would be expected for simple T-cell removal. In vitro assays on enriched neutrophil preparations revealed that 80-95% of these cells were reactive with ACM-1 and 9AE10. Thus, it appears that in the rabbit, T cells and neutrophils share a major epitope.     

Complexes of disubstituted benzene positional isomers with α-cyclodextrin

Stability constants for complex formation between α-cyclodextrin and some ortho-, meta-, and para-disubstituted benzenes were measured in aqueous solution at 25°C by potentiometry, spectrophotometry, competitive spectrophotometry, and solubility. All systems form 1:1 complexes, some para substrates form 1:2 complexes (one substrate to two cyclodextrins), but no meta substrates form 1:2 complexes. Ortho substrates form weak complexes. These observations are accounted for in terms of a binding site molecular model. On the average over many systems, K₁₁ (para) and K₁₁ (meta) are approximately equal. Major discrepancies (greater than a factor of two) are diagnostic of significantly different electronic or steric effects in the complexing abilities of the isomeric substrates.     

Light-dependent chemical modification of thylakoid membrane proteins with carboxyl-directed reagents

Spinach chloroplast thylakoid membranes were chemically modified with membrane penetrating reagents reactive toward protein carboxyl groups, a carbodiimide and the nucleophiles [¹⁴C]glycine ethyl ester or [³H]serotonin. The reagents, being weak bases, were accumulated within the inner aqueous space in the light, due to the low pH inside. Both the accumulation and the low pH stimulating effect on the carbodiimide activation step contributed to a greater labeling in the light compared to dark, and uncouplers inhibited most of the light-dependent increase. Hence, it is likely that the proteins showing the light-dependent, uncoupler-sensitive labeling have those parts located within the inner aqueous space or within the membrane itself. While many membrane proteins which separated on sodium dodecyl sulfate-polyacrylamide gels (12.5–25% gradient) showed some increased labeling in the light, the most conspicuous were the four polypeptides of the chlorophyll $\text{a}\text{b}$ light-harvesting complex. The light-harvesting complex was purified from dark- and light-treated, labeled membranes. The resultant preparation showed about a sixfold, light-dependent, uncoupler-sensitive labeling increase compared to dark conditions. Polypeptides near 6 and 8 kdalton showed light-dependent, uncoupler-resistent increases in carboxyl group modification, which could be due to localized acidic conditions near sites of proton release.     

Alteration of thermal stability of glucose oxidase associated with the redox states

By the method of differential scanning calorimetry, it was found that thermal stability of glucose oxidase was dependent on its redox states. The oxidized form showed an apparent denaturation temperature at 76°C and the denaturation enthalpy was approximately 865 kcal/mol. On reduction of the enzyme, the denaturation temperature increased by about 10°, but no significant change was seen in the denaturation enthalpy. The activation energies of the denaturation of the oxidized and the reduced enzymes were about 89 and 103 kcal/mol, respectively. These results may imply conformational changes in the catalytic turnover of this enzyme.     

Immunocapillarymigration with enzyme-labeled antibodies: Rapid quantification of C-reactive protein in human plasma

Various procedures to improve the sensitivity and precision of antigen quantitation by immunocapillarymigration are investigated. The best results are obtained when using porous strips of cellulose acetate with covalently attached antibodies and when enzyme-labeled antibodies are used to expose the antigen-covered areas of the strips. Such a system has a sensitivity of 0.15 mg/liter and a precision of 7%. It allows a rapid quantitation of human C-reactive protein without the use of laboratory instrumentation.     

Cytochrome c interaction with membranes. I. Use of a fluorescent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes

Quenching of 12-(9-anthroyl) stearic acid (AS) fluorescence by cytochrome c occurs through an energy-transfer mechanism and can be used to measure the binding of the cytochrome to artificial and mitochondrial membranes. The quenching of AS³ fluorescence is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ below 25 msec and above 500 msec) and its extent diminishes at high salt concentration or at high pH and increases in the presence of negatively charged lipids.Addition of cytochrome c to cytochrome c-depleted mitochondria results in binding of the cytochrome to the membrane and quenching of AS fluorescence. The affinity of oxidized cytochrome c for cytochrome c-depleted mitochondria is 1.8 × 10⁶m, while the affinity constant for reduced cytochrome c is 0.5 × 10⁶m. The lower affinity of the reduced cytochrome c for mitochondrial membranes is in accordance with midpoint potential differences between the bound and free forms.     

Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide

A convenient method for postcolumn carbohydrate labeling has been developed. Eluates of borate complex anion-exchange columns are mixed with a reagent solution prepared from an aqueous solution of 2-cyanoacetamide and a borate buffer (pH 10.5), and the mixture is heated in a 10-m reaction coil at 100°C. Measurement of the absorbance of the product at 276 nm permits high reproducibility determination of 5 to 500 nmol of aldoses. Some carbonyl compounds are positive to this reaction, but most do not interfere with the analysis because their peaks do not appear in the aldose region. Ascorbate gives a small peak between those of mannose and fucose, but interference is negligible for equimolar amounts of ascorbate and these aldoses. This method is applied to and gives satisfactory results in the analysis of monosaccharides from various types of glycoconjugates.     

Measurement of aspartylglucosamine in physiological fluids with an amino acid analyzer: Fused peak analysis with dual photometers

Methods for the quantitation of aspartylglucosamine using an amino acid analyzer equipped with a physiological column and dual photometers were developed. Aspartylglucosamine and urea coelute, but their ninhydrin derivatives have different color factors at 440 and 590 nm. The concentration of aspartylglucosamine may be determined in a urea mixture by amino acid chromatography, absolute peak area measurement, and the 590:440 peak area ratio. A second method was also developed in which urea was completely digested with urease before chromatography, permitting the direct quantitation of aspartylglucosamine.