The excitation of the heart and its modification under the influence of the chemical mediators and the cardiac nerves: II. A particular case of the one-factor theory

Previous work (Macey, 1952) in the application of the one-factor theory to the heart is extended. The rate of production of the excitatory state is assumed to be linear. Two possible mechanisms are indicated whereby such a situation might arise. Assumptions are made regarding the mode of action of the chemical mediators on the heart, and an equation is derived relating the heart rate to the frequency of nerve impulses traveling along the cardiac nerves. This result compares favorably with the experimental findings of A. Rosenblueth and F. A. Simeone (1934). Other experimental results are interpreted in terms of the theory. United States Public Health Service Fellow in Mathematical Biology.     

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide.

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate alpha-N-benzoyl-L-arginine ethl ester were studied in the region of pH 3.5-9.0. All substrates (and inhibitors) test, with the exception of alpha-N-benzyoyl-L-arginine p-nitroanilide, displayed approximately a two fold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is probably Trp-177.     

The mechanism of the chemical synthesis of oligonucleotides and its synthetic consequences.

The data obtained mainly by pulsed NMR spectroscopy on phosphorus nuclei on the mechanism of the internucleotide phosphodiester (PDE) group formation are summarised. With arylsulphonyl chloride as condensing reagent monomeric nucleotide derivative B (nucleoside metaphosphate or its pyridinium adduct) is the highly reactive intermediate. In the presence of PDE groups in nucleoside or nucleotide component the significantly less reactive derivatives with trisubstituted pyrophosphoryl residues are formed both with arylsulphonyl chloride and dicyclohexylcarbodiimide (DCC). The reactive B form of nucleotide component may be obtained using greater excess of arylsulphonyl chloride with simultaneous convertion of PDE groups to tetrasubstituted pyrophosphates amenable to side reactions. The convertion of PDE groups to easily hydrolysable dicyclohexylurea derivatives by reaction with DCC is proposed to reversible blocking of PDE groups of nucleoside component. The B type derivatives of mononucleotides or oligonucleotides with blocked PDE groups seems to be the best nucleotide components.     

Physical consequences of chemical modification: circular dichroism of the kethoxalated oligoribonucleotides

Alterations in CD spectra are found in G-containing oligoribonucleotides after modification with kethoxal (beta-ethoxy--alpha-ketobutyraldehyde). Stacking interactions in kethoxalated oligomers are followed by temperature dependence of their CD amplitudes. It is shown that for oligomers with nucleosides in anti-conformation adduct formation destroys the stacking interaction with 3'-neighbour but not with a 5'-neighbour. For nucleosides in non-standard conformation (i.e. syn-conformation of guanine in GpGpCp) the physical alteractions may be seen in those cases, when the substituting group affects the initial conformation or the interplane base contacts via, for instance, blocking NH(2)-group of guanine in GpUp.The results demonstrated that even a single monomer modification in a polymer chain could not be considered as a local event having no influence on the three-dimensional structure. The degree of conformational disorders depends both on the conformation of single nucleotides in the stack and on the nature of the nearest neighbours of the modified base.     

Ca2+ transport in the sarcolemma of the myometrium under its chemical modification

The effect of some chemical modifying agents of the sarcolemma surface--dicyclohexicarbodiimide, trinitrobenzolsulphuric acid, dithiotreitol and nitrit-anions on passive transsarcolemmic accumulation of Ca2+ by the pig myometrium sarcolemma property oriented vesicules was studied. The comparative analysis of these substances action under proton transmembrane gradient dispersion is presented. The significance of surface amino-, carboxy groups and disulphide membrane bounds in Ca2+ transport mechanisms as a corresponding aspect to some contemporary ideas about Ca(2+)-channel has been defined. The hypothesis is made that the proton transmembrane gradient dissipation leads to dislocation of carboxyl groups and disulphid bounds in relation to the membrane surface resulting in increasing Ca(2+)-permeability of the sarcolemma. The increase of sarcolemma permeability for Ca2+ under the action of 10 nM NO-2 was demonstrated. On the base of experimental data the supposition was made that NO-2 modified amino- and carboxylate groups of the sarcolemma surface. Under the chemical modification of these groups the nitrit-anions inhibit transsarcolemmic Ca2+ movement. NO-2 is not a result of sarcolemma surface SH-groups oxidation, while possibly NO2- protects some functionally important disulphide bridges of Ca(2+)-channel from their chemical restoration.     

Cysteine in the manganous-isocitrate binding site of pig heart TPN-specific isocitrate dehydrogenase: I. Kinetics of chemical modification and properties of thiocyano enzyme

Pig heart TPN-dependent isocitrate dehydrogenase is inactivated by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB). The dependence of the rate constant for inactivation on the reagent concentration is nonlinear, and can be analyzed in terms of the existence of two mechanisms for reaction with the enzyme, one involving reversible binding prior to inactivation and the other a bimolecular reaction. Cyanide reacts with the inactive modified enzyme to yield thiocyano-isocitrate dehydrogenase without increasing the catalytic activity; this result suggests that inactivation by DTNB is not due to steric hindrance by the bulky thionitrobenzoate group bound to the enzyme. The inactive thiocyano enzyme binds manganous ion normally. In contrast to its effect on native enzyme, however, isocitrate does not strengthen the binding of Mn²⁺ to the thiocyano enzyme; the tightened binding of manganous-isocitrate may be critical for the catalytic activity of the enzyme. Protection against inactivation by DTNB is provided by isocitrate plus the activator, manganous ion, or the competitive inhibitor, calcium ion. The concerted inhibitors oxalacetate and glyoxylate, when present together with Mn²⁺ and TPN, also protect against loss of activity. A marked decrease in the inactivation rate constant to a finite limiting value is caused by saturating concentrations of TPNH and Mn²⁺, indicating that these ligands do not bind directly at the sites attacked by DTNB. The number of cysteine residues which react with DTNB concomitant with inactivation depends on the ligands present in the reaction mixture. In all cases, the equivalent of one -SH reacts without affecting activity. In the presence of Mn²⁺ and α-ketoglutarate, which do not appreciably affect the inactivation rate, loss of activity is proportional to reaction with two -SH groups. These results suggest that the integrity of a maximum of two cysteine residues is essential for the function of the pig heart isocitrate dehydrogenase, and that at least one cysteine residue may be located within the manganous-isocitrate binding site.