Nucleotide specificity of succinate thiokinases from bacteria.

The nucleotide specificity of succinate thiokinases, isolated from Escherichia coli, Aerobacter aerogenes, and Pseudomonas citronellolis, was determined and found to be nonspecific for adenine and 6-oxopurine nucleotides, guanine, and hypoxanthine. The enzyme from Herellae vaginicola was specific for the 6-oxopurine nucleotides. Succinate thiokinases from E. coli, A. aerogenes, and P. citronellolis also demonstrated purine nucleoside diphosphokinase activity (P-NDPK), which was 4, 9, and 40%, respectively, of the succinate thiokinase activity. P-NDPK activity was slightly stimulated by coenzyme A (CoA) and slightly inhibited by succinate; in the presence of both CoA and succinate, P-NDPK activity increased three-, three-, and sevenfold for the E. coli, A. aerogenes, and P. citronellolis enzymes, respectively. Isoelectric focusing demonstrated multiple forms of each enzyme, and the molecular weights of the A. aerogenes, P. citronellolis, and H. vaginicola enzymes were approximately 155,000.     

Purification and specificity of antibodies to adenosine.

Antibodies to adenosine were elicited in rabbits by immunization with bovine serum albumin-adenosine conjugate. The antibodies were purified and fractionated on two affinity columns (Sepharose-oligo(A) and Sepharose-AMP). Two families of antibodies have been obtained. The antibodies purified on the Sepharose-oligo(A) column react with poly(A) while those purified on the Sepharose-AMP column do not, as shown by gel diffusion. The association constants for the binding of Fab fragments or IgG purified on the Sepharose-oligo(A) column and several haptens were deduced from dialysis equilibrium, fluorescence quenching and displacement of AMP-fluorescein conjugate. The antibodies mainly recognize adenine, and the ribose or the phosphate group of (or AMP derivatives) do not play a critical role in the interaction. Thermodynamic parameters for adenosine-Fab fragments complexes have been determined deltaH degrees = 16 kcal/mole and deltaS degrees = - 15 cal. degree-1 mole-1. Circular dichroism studies indicate that about three nucleotide residues penetrate the binding site of Fab fragments.     

Characterization of transferrin binding and specificity of the placental transferrin receptor

This study systematically examined the characteristics of specific binding of adult diferric transferrin to its receptor using a Triton X-100 solubilized preparation from human placentas as the receptor source. The following information was obtained. The ionic strength for maximal binding is in the range of 0.1-0.3 M NaCl. The pH optimum for specific binding extends over the range, from pH 6.0-10.0. Specific binding of diferric transferrin is not affected by 2.5 approximately 50 mM CaCl2 or by 10 mM EDTA. Triton X-100 in the concentration range of 0.02-3.0% does not affect specific binding. Specific binding is saturated within 10 min at 25 or 37 degrees C in the presence of excess amounts of diferric transferrin. The binding is reversible and the dissociation of diferric transferrin from the transferrin receptor is complete within 40 min at 25 degrees C. Apotransferrin, both adult and fetal, showed less binding than the holotransferrin species by competitive binding assay in the presence of 10 mM EDTA independent of up to 20 mM CaCl2. A 1500-fold molar excess of adult and fetal apotransferrin is required to give 40% inhibition for 125I-labeled diferric transferrin binding. Since calcium ion is not a factor, and since apotransferrin has such high binding affinity for iron (Ka = 1 X 10(24], this experiment suggests that the EDTA was necessary to prevent conversion of apotransferrin to holotransferrin from available iron in the reaction system. The specificity of the transferrin receptor for transferrin was examined by competitive binding studies in which 125I-diferric transferrin binding was measured in the presence of a series of other proteins. The proteins tested in the competitive binding studies were classified into three groups; in the first group were human serum albumin and ovalbumin; in the second group were proteins containing iron ions, such as hemoglobin, hemoglobin-haptoglobin complex, heme-hemopexin complex, ferritin, and diferric lactoferrin; in the third group were the metal-binding serum proteins, ceruloplasmin and metallothionein. None of these proteins except ferritin showed inhibition of diferric transferrin binding to the receptor. The effect of ferritin was small since a 700- to 1500-fold molar excess of ferritin is required for 50% inhibition of binding of diferric transferrin to the receptor.     

Purification and specificity of antibodies to inosine 5'-monophosphate

Antibodies to inosine 5'-monophosphate elicited in rabbits by immunization with a conjugate of IMP (oxidized with periodate) and bovine serum albumin have been purified by affinity chromatography. By the use of two affinity columns, Sepharose-IMP and Sepharose-oligo(I), the antibodies have been fractionated into three fractions. By gel diffusion, the three fractions were found to react with the conjugates of bovine serum albumin and IMP, GMP and AMP respectively. The association constants for the binding of the Fab fragments purified on the Sepharose-oligo(I) column and several haptens have been deduced from fluorescence experiments. It is shown that the base and the phosphate group play an important part in the binding of IMP to Fab fragments. No reaction has been found between the antibodies and poly(I).poly(C) by gel diffusion. However, the antibodies interact with poly(I).poly(C) since they decrease the thermal stability of poly(I).poly(C).     

Glycosyl transfers from UDP-sugars to lipids of plant membranes: identification and specificity of the transferases

Microsome preparations extracted from wheat roots or sycamore cell suspensions catalyzed the transfer of sugar from nucleotide-sugars to endogenous lipidic acceptors. The nature of the products biosynthesized from UDP-Glc, GDP-Glc, UDP-Gal, UDP-Xyl or UDP-Arab was examined. Sterylglycosides were obtained from UDP-Gglc, GDP-Glc or UDP-Xyl. Galactosyldiglycerides were synthesized from UDP-Gal. When UDP-Glc or UDP-Gal was used as a substrate, a membrane-bound 4-epimerase interconverted the epimeric nucleotide-sugars, thereby allowing the simultaneous biosynthesis of galactosyldiglycerides and sterylglucosides. The biosynthesis of free and acylated sterylglucosides from UDP-Glc, without interference of other glycosyl transfer reactions, was obtained by the omission of Mg++ ions from the incubation medium. The biosynthesis of galactosyldiglycerides from UDP-Gal without interference of other transfer reactions was obtained when digitonin was added to the incubation medium of sycamore microsomes.     

Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.     

Proteolytic enzymes as probes for the triple-helical conformation of procollagen

Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation.     

Glyconamides as inhibitors of human beta-glucosidases and beta-galactosidases.

d-Gluconamide, d-gluconyl hydrazide, and N-(6-aminohexyl)-d-gluconamide were prepared from d-glucono-1,5-lactone by treatment with ammonia, hydrazine, and 1,6-diaminohexane, respectively. These d-gluconamide derivatives were tested for their inhibitory action on human liver lysosomal glucocerebrosidase and human spleen neutral aryl β-glucosidase. Analogous d-galactonamide derivatives were evaluated for their inhibition of human spleen galactocerebrosidase and G_M1-ganglioside β-galactosidase. d-Gluconyl hydrazide and d-gluconamide were effective inhibitors of the lysosomal glucocerebrosidase, attaining 50% inhibition at 5 and 12 mm, respectively. In contrast, N-(6-aminohexyl)-d-gluconamide did not inhibit the glucocerebrosidase. d-Gluconyl hydrazide was also the most effective inhibitor of human liver and spleen aryl β-glucosidase, 50% inhibition being achieved at 4 mm concentration (competitive inhibition, K_i = 0.4–0.9 mM). d-Galactonamide was the most effective inhibitor of spleen galactocerebrosidase; 4 mm d-galactonamide caused 50% inhibition of the enzyme activity (noncompetitive inhibition). N-(6-Aminohexyl)-d-galactonamide is a potent inhibitor (90% inhibition, 5 mm) of G_M1-ganglioside β-galactosidase but is without effect on galactocerebrosidase. It has, therefore, the potential usefulness in distinguishing between two of the galactosphingolipid β-galactosidases.     

Dissociated limb bud cells of chick embryos can express lens specificity when reaggregated and cultured in vitro

Limb bud cells of chick embryos (stages 23–24) were dissociated into single cells, reaggregated, and cultured in vitro for about a week. δ-Crystallin, generally thought to be a lens-specific protein in the chick, was detected in the aggregates by indirect immunofluorescent staining, double immunodiffusion test, and immunoelectrophoresis with specific antiserum against δ-crystallin. Cells containing δ-crystallin were distributed in epidermal cell clusters and also in mesenchymal tissues surrounding cartilage nodules in the aggregates. Those cells in mesenchymal tissues were shown to have originated from the mesoderm of the limb bud, and those in epidermal cell clusters probably originated from the ectoderm. The possible cellular origin of this appearance of δ-crystallin was discussed.     

A specific DNA orientation in the filamentous bacteriophage fd as probed by psoralen crosslinking and electron microscopy.

The molecular structure of the single-stranded fd DNA inside its filamentous virion has been stabilized by the photochemical reaction with a psoralen derivative and examined in the electron microscope. The results support the notion that the 6389 nucleotide-long DNA molecule is folded back on itself inside the 1 μm-long protein coat. At one end of the virion, there exists a DNA hairpin region 200±50 base-pairs long. This “end hairpin” is mapped on the fd genome to the site of the replication origin. The most stable in vitro hairpin of fd DNA has been mapped previously to this same site. This unique duplex region of fd DNA may play an important role in the formation of specific protein-DNA complexes which are crucial to stages of the fd life cycle: the adsorption of the phage to the bacteria, the initiation of replication of the single-stranded DNA, and the assembly of newly synthesized DNA strands into the filamentous virions.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.     

Tissue specificity of 3′-untranslated sequence of myosin light chain gene: Unexpected interspecies homology with repetitive DNA

Using the 3′ noncoding and coding sequences of chick heart myosin light chain mRNA cloned into Escherichia coli as probes, it was observed that, while the coding sequence shared homology with myosin light-chain mRNAs from other sources, the 3′ noncoding sequence was specific for chick heart muscle. This property was used to detect chick heart-specific myosin light-chain gene activity in chick blastoderms of very early developmental stages where cells of different muscle origins cannot be distinguished morphologically. However, in spite of the tissue-specific divergence of the 3′ noncoding sequence of myosin light-chain gene, which is present in a single copy in the chick genome, a surprising homology with DNA from such a diverse source like Dictyostelium discoideum was noted. The sequence homologous to chick myosin light-chain DNA was apparently present in a high repetition frequency in the Dictyostelium genome.     

O-methylhydroxylamine as a reagent for NAD+ modification in urocanase.

The inhibition of urocanase from Pseudomonas putida by O-methylhydroxylamine has been characterized as being due to the formation of an adduct between CH₃ONH₂ and NAD⁺, the latter of which has been recently shown to be a tightly bound coenzyme for this urocanase. Inhibition is maximal at pH 8.5 and is blocked by the presence of the substrate analog imidazole propionate. Loss of catalytic activity corresponds directly with the binding of 1 mol of ¹⁴CH₃ONH₂ per mole of enzyme, and partial reversibility of the modification, achieved by dialysis at pH 7.5, is accompanied by concomitant restoration of enzymatic activity. No incorporation of ¹⁴CH₃ONH₂ into urocanase is seen when enzyme-bound NAD⁺ is first converted to NADH or when NAD⁺ is removed by prior treatment of urocanase with 8 m urea. Stability and spectral properties of the CH₃ONH · NAD adduct are consistent with previous data reported for the product of the hydroxylamine reaction with NAD⁺. It is concluded that other urocanases which exhibit inhibition by hydroxylamine may likewise contain NAD⁺ as an essential coenzyme and that the use of ¹⁴CH₃ONH₂ as a reversible modification reagent for NAD⁺ should prove helpful for studies on the role of NAD⁺ in the urocanase catalytic process.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

3,4,5,3',4'-Pentachlorobiphenyl as a useful inducer for purification of rat liver microsomal cytochrome P448.

An easy purification of rat liver microsomal cytochrome P448 was performed by using 3,4,5,3′,4′-pentachlorobiphenyl as an inducer. The cytochrome P448, a high spin form, was purified to 18.1 nmoles/mg protein with a good yield by ω-aminooctyl Sepharose 4B column chromatography followed by a hydroxyapatite column chromatography. This hemoprotein cross-reacted with antibody to cytochrome P448 from β-naphthoflavone-treated rats, but not with antibody to cytochrome P450 from phenobarbital-treated rats at all. The results of amino acid analyses suggested that this cytochrome P448 is similar to cytochrome P448 of 3-methylcholanthrene-treated rats.