Controlled coupling of mildly reduced proteins to Sepharose gelatin by heterobifunctional reagent

The heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate was utilized for controlled coupling of mildly reduced BSA and lysozyme to Sepharose gelatin to prepare immunoabsorbents. Each reaction step was examined and quantitated. The free amino groups on gelatin after coupling gelatin to cyanogen bromide-activated Sepharose as well as the number of 3-(2-pyridyldithio) propionyl groups on Sepharose gelatin were quantitated. Coupling of mildly reduced BSA as well as lysozyme to PDP-Sepharose gelatin occurred through sulfhydryl-disulfide exchange and permitted the formation of an immunoabsorbent. The immunoabsorbents were capable of binding the respective antibody and the eluted antibodies were pure and free of plasma proteins.     

A versatile primer for DNA sequencing in the M13mp2 cloning system

A primer for DNA sequencing by the chain-termination method in the M13mp2 cloning system was constructed and amplified. The primer was isolated as an EcoRI/AluI restriction fragment. After conversion of the AluI end into an EcoRI end the fragment was cloned in pBR325 from which it can be recovered by cleavage with EcoRI. The primer hybridizes to the single-stranded DNA of the mature M13mp2 phage next to the site of insertion thereby directing DNA synthesis along the inserted DNA.     

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and −H4) isozymes

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M₄ = muscle type; LDH-H₄ = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M₄ or LDH-H₄. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M₄ and ≈2700 for the H₄ inhibitor were calculated. An apparent K_i of ≈3 × 10^?5 mM for the H₄ and ≈7 × 10^?5 mM for the H₄ inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M₄-specific) and 2.4 (LDH-H₄-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k₁ for the dimerisation step of M₄ = 2.0 × 10⁵ M^?1 · s^?1 and of H₄ = 8.2 × 10⁴ M^?1 · s^?1; k₂ for the tetramerisation step of M₄ = 2.8 × 10⁵ M^?1 · s^?1 and of H₄ = 1.2 × 10⁵ · M^?1 · s^?1, were calculated, the second step still being the faster one.     

Glycine and glyoxylate decarboxylation in Nicotiana rustica roots

Glycine was decarboxylated only by intact mitochondria to yield carbon dioxide, formaldehyde, and ammonia, probably present as pyridoxamine phosphate. The formaldehyde could become incorporated into serine, via N⁵N¹⁰ methylene-FH₄, and a requirement was demonstrated for pyridoxal phosphate. Similarly, glyoxylate with pyridoxamine phosphate was also decarboxylated to formaldehyde and carbon dioxide. Glyoxylate could be decarboxylated by at least two additional pathways. One consisted of oxidative decarboxylation yielding formate and carbon dioxide, and requiring thiamine pyrophosphate, manganese ions, and oxygen. The other consisted of glyoxylate condensation with 2-oxoglutarate, yielding carbon dioxide and an intermediate which, upon decarboxylation, appeared to be hydroxylevulinic acid.     

Immunoglobulin recognition of synthetic and natural left-handed Z DNA conformations and sequences

The relative immunogenicities of the poly[d(G-C)] and poly[d(A-C) · d(G-T)] families of helices have been determined. The specificities of the resultant immunoglobulins have been characterized for recognition of different synthetic and natural left-handed sequences and conformations. Certain modifications of poly[d(G-C)] in the sugar-phosphate bacbone and cytosine C-5 potentiate the right(R)-to-left(L) (B → Z) transition under physiological conditions. The resulting polynucleotides, poly[d(G^S-C)], poly[d(G-io⁵C)], poly[d(G-br⁵C)] and poly[d(G-m⁵C)], are also highly immunogenic. In contrast, DNAs incapable of assuming the left-handed conformation under physiological salt concentrations are weakly or non-immunogenic. These include unmodified poly[d(G-C)] as well as members of the poly[d(A-C) · d(G-T)] family of sequences bearing pyrimidine C-5 substitutions (methyl, bromo, iodo). These polynucleotides undergo the R → L isomerization under more stringent ionic and thermal conditions.The specificities of purified polyclonal and monoclonal anti-Z DNA immunoglobulins (IgG) were measured by binding to radiolabeled polynucleotides, by electrophoretic analysis of IgG bound to covalent closed circular DNAs, and by immunofluorescent staining of polytene chromosomes. The salt-induced left-handed forms of poly[d(G-C)] and its derivatives (including the cytidine C-5 methyl, bromo, iodo, and N-5 aza substituted polynucleotides) and of the modified poly[d(A-C) · d(G-T)] polymers are bound to varying degrees by different antibodies. The patterns of substrate recognition demonstrate the existence of several antigenic domains in left-handed DNAs, including the helix convex surface and the sugar-phosphate backbone. Substitutions in these regions can produce enhancing (required substitutions), neutral, or inhibitory effects on subsequent IgG binding. Additionally, certain modifications of either the convex surface of Z DNA at the C-5 position of cytidine (i.e. a methyl group) or of the backbone (i.e. phosphorothioate substitution) can lead to polymorphic lefthanded conformations that are compatible with antibody binding when present individually but not in combination. The recognition patterns exhibited with DNA substrates from the two DNA families indicate that some, but not all, IgGs show specificity for different nucleotide sequences.The anti-Z DNA IgGs were used to probe for specific left-handed Z DNA determinants on plasmid (e.g. pBR322) or viral (e.g. simian virus 40 (SV40)) DNAs and on the acid-fixed polytene chromosomes of dipteran larvae. At their extracted superhelical density, the negatively supercoiled form I, but not the relaxed, nicked, or linear forms of all tested plasmid and viral DNAs specifically bind sequence-independent anti-Z IgGs. Dimers, trimers and higher oligomers of form I DNA cross-linked by bivalent anti-Z IgGs are formed with numerous (e.g. φX174, SV40, pBR322) genomes. Their occurrence depends upon IgG concentration and specificity, the conditions of ionic strength and temperatures and the DNA genome. The IgG cross-linked DNA multimers are converted to monomers by dithiothreitol reduction. Sequence-independent monovalent anti-Z Fab fragments bind form I DNA but do not generate oligomeric species. Multimers of order >2 indicate the existence of at least two anti-Z Ig binding sites per molecule, as in the case of SV40. IgGs differ in their ability to form stable complexes with some sites on natural DNAs, presumably due to their sequence and conformation binding specificities. A differential binding of these antibodies is also observed in certain bands of polytene chromosomes, such as the telomeric regions that are involved in chromosome associations.     

The inactivation of thymidylate synthase by periodate

Thymidylate synthase from methotrexate-resistant Lactobacillus casei rapidly lost about 90% of its catalytic activity when incubated with an equimolar concentration of IO4- at 0 degree C. Nearly complete inhibition resulted when the IO4- concentration was twice the enzyme concentration or higher. The inhibition reaction appeared to be pseudo-first-order with respect to enzyme when IO4- was in excess. The substrate dUMP, the product dTMP, and inorganic phosphate all protected the enzyme from inactivation by IO4-, with the order of effectiveness: dUMP greater than dTMP greater than phosphate. Deoxyuridine, which is not a substrate, did not protect the enzyme. Titrations with dithiobis(2-nitrobenzoate) (DTNB) showed that approximately 1.5 titratable SH groups were lost when thymidylate synthase was completely inhibited by IO4-. Essentially no reactivation occurred when periodate-inhibited enzyme was dialyzed against buffered 2-mercaptoethanol (ME) or dithiothreitol (DTT). Enzyme that had been treated with p-hydroxymercuribenzoate, DTNB, or methylmethanethiosulfonate prior to treatment with periodate could be completely reactivated with ME or DTT.     

Mechanism of inhibition of rat brain (Na +-K +)-stimulated adenosine triphosphatase reaction by cadmium and methyl mercury

The mechanisms of inhibition of rat brain Na ⁺-K ⁺- ATPase by cadmium chloride (CdCl₂) and methylmercuric chloride (CH₃HgCl) were studied in vitro by assessing the effects of these heavy metals on this enzyme and associated component parameters. Both the heavy metals significantly inhibited the overall Na ⁺-K ⁺ -ATPase in a concentration-dependent manner with an estimated median inhibitory concentration (IC-50) of 3.2 × 10^?5M for CdCl₂ and 6 × 10^?6M for CH₃HgCl. Protection of enzyme against heavy metal inhibition by 5 × 10^?5M to 1 × 10^?4 M dithiothreitol (DTT) and glutathione (GSH) or cysteine (CST) indicates that both monothiols and dithiols have the same ability in regenerating sulfhydryl (–SH) groups or chelating the metals. Inhibition of K⁺-p-nitrophenyl phosphatase (K⁺-PNPPase), the component enzyme catalyzing the K⁺-dependent dephosphorylation in the overall Na ⁺-K ⁺ATPase reaction by these heavy metals, indicates that the mechanism of inhibition involves binding to this phosphatase. Reversal of K⁺-PNPPase inhibition by DTT, GSH, and CST suggests sulfhydryl groups as binding sites. Binding of ³H-oubain, a cardiac glycocide and inhibitor of both phosphorylation and dephosphorylation, to brain fraction was significantly decreased by CH₃HgCl, and this inhibition was reversed by the three thiol compounds, suggesting presence of –SH group(s) in the ouabain receptor site. Cadmium chloride failed to inhibit the binding of this receptor, indicating that the mechanics of inhibition of ATPase by CH₃HgCl and CdCl₂ are different from each other. The results suggest that the critical conformational property of enzyme common to both kinase (E₁) and phosphatase (E₂) is susceptible to CH₃HgCl whereas only phosphatase is sensitive to CdCl₂.     

Na+/Ca2+ exchange of isolated sarcolemmal membrane: effects of insulin,oxidants and insulin deficiency

In this study we prepared sarcolemmal fractions from bovine and rat hearts; their Na⁺K⁺ ATPase activities, measured in the presence of saponin to unmask latent Na⁺K⁺ ATPase, were 59.4 and 48.8 µ mol Pi/mg protein · h, respectively. The rate of Na⁺dependent Ca2⁺ uptake was linear for the first 10 s and a plateau was reached in 3 min. Oxidation by free radical generation either with H₂O₂, FeSO₄ plus DTT or xanthine oxidase plus hypoxanthine stimulated Na⁺/Ca²⁺ exchange in a time-dependent manner. The stimulation was abolished by deferoxamine or o-phenanthroline. By contrast, oxidation by HOCI inhibited Na⁺/Ca²⁺ exchange in proportion to its concentration, and this inhibition was antagonized by DTT. DTT alone had no effect on the exchange. Insulin stimulated Na⁺/Ca²⁺ exchange, its maximal effect was attained after 30min incubation with 100 µ units/ml. N-ethylmaleimide inhibited the exchange both in the presence and in the absence of insulin. Sarcolemmal fractions prepared from hearts of alloxan-treated, acutely diabetic rats showed a significant decrease in Na⁺/Ca²⁺ exchange. Addition of insulin in vitro significantly stimulated Na⁺/Ca²⁺ exchange of both diabetic and control groups. The results indicate that sarcolemmal Na⁺/Ca²⁺ exchange function is modulated by oxidation-reduction states and by the presence of insulin.