Proton transfer and polarizability of hydrogen bonds in proteins coupled with conformational changes. I. Infrared investigation of poly(glutamic acid) with various N Bases

The nature of hydrogen bonds formed between carboxylic acid residues and histidine residues in proteins is studied by ir spectroscopy. Poly(glutamic acid) [(Glu)_n] is investigated with various monomer N bases. The position of the proton transfer equilibrium OH…?N ? O^?…?H⁺N is determined considering the bands of the carboxylic group. It is shown that largely symmetrical double minimum energy surfaces are present in the OH…?N ? O^?…?H⁺N bonds when the pK_a of the protonated N base is two values larger than that of the carboxylic groups of (Glu)_n. Hence OH…?N ? O^?…?H⁺N bonds between glutamic and aspartic acid residues and histidine residues in proteins may be easily polarizable proton transfer hydrogen bonds. The polarizability of these bonds is one to two orders of magnitude larger than usual electron polarizabilities; therefore, these bonds strongly interact with their environment. It is demonstrated that water molecules shift these proton transfer equilibria in favor of the polar proton boundary structure. The access of water molecules to such bonds in proteins and therefore the position of this proton transfer equilibrium is dependent on conformation. The amide bands show that (Glu)_n is α-helical with all systems. The only exception is the (Glu)_n-n-propylamine system. When this system is hydrated (Glu)_n is α-helical, too. When it is dried, however, (Glu)_n forms antiparallel β-structure. This conformational transition, dependent on degree of hydration, is reversible. An excess of n-propylamine has the same effect on conformation as hydration.     

Significance of binary and ternary copper(II) complexes for the promotion and protection of adenosine 5′-di- and triphosphate toward hydrolysis

The dephosphorylation of ADP and ATP was characterized as the first-order rate constant in dependence on pH in the absence and presence of Cu²⁺, and together with Cu²⁺ and a second ligand. The reaction is strongly accelerated by Cu²⁺ and passes through pH optima at about 6.2 and 6.5 for the Cu²⁺ ?ADP and ?ATP systems, respectively (I = 0.1, NaClO₄; 50°C). In the presence of 2,2′-bipyridyl (Bipy), ternary complexes are formed with the nucleotides ADP or ATP (NP), Cu(Bipy)(NP), which are very stable towards dephosphorylation over a large pH range. Similar stabilizing effects were observed in ternary complexes formed with imidazole or OH^?. These results can easily be rationalized by taking into account that in the binary Cu²⁺ complexes macrochelates are formed by the interaction between the adenine moiety and the metal ion. This interaction is crucial for obtaining the labile species and hence, in the mixed-ligand complexes, where the macrophelate can not be formed, the phosphates are protected toward hydrolysis. In agreement with these results is the dephosphorylation behavior of Cu(CDP)^? and Cu(CTP)^2?; they are rather stable. This is in accord with the small coordination tendency of the cytosine moiety.By computing the pH dependence of the distribution of the several species, it is shown that the active species are Cu(ATP)^2? and Cu(ADP)^? and not the hydroxy complexes, [Cu(ATP)(OH)]₂^6? and [Cu(ADP)(OH)₂^4? as were suggested earlier. With the aid of the initial rate, $\text{ν}{}_0\text{=}\text{d}\text{[}\text{PO}{}_4{}^{\mathrm{3?}}\text{]}\text{d}\text{t}$, the rate laws of the ascending side of the pH optima were determined: $\text{ν}{}_0\text{= k}\text{[}\text{Cu(NP)}\text{]}\text{[}\text{H}{}^{\mathrm{+}}\text{]}$. The descending side of the pH optima is attributed to the formation of Cu(NP)(OH), where the metal ion interaction with N-7 of the adenine moiety is inhibited.     

Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis.

The structure of carp muscle calcium-binding parvalbumin has been refined to an overall residual, ($\text{Σ¦F}{}_0\text{− F}{}_{\mathrm{c}}\text{¦}\text{Σ¦F}{}_0\text{¦}$), of 0.25 by a combination of model building and difference Fourier analyses. The atomic positions were allowed to vary up to 0.25 Å from their idealized positions; individual temperature factors ranged from 2 to 150; and 138 solvent peaks were included in this final structure factor calculation. The effects of varying these parameters were analyzed. The treatment of low-order reflections and the influence of solvent have been analyzed in terms of Babinet's principle. These procedures and results are applicable to other protein refinement problems. The errors in co-ordinates are estimated to range from 0.15 Å for the Ca²⁺ ions to 0.30 Å for internal side chain atoms.A van der Waals' radii study indicates that 42% of the crystal volume is occupied by solvent. There are 16 intermolecular contacts. Of the 108 main chain peptide hydrogen atoms, 15 are neither in contact with solvent nor involved in hydrogen bonds. These “lost” hydrogen bonds are considered to be important to the proposed model of function.     

Some dilute-solution parameters of the levan of streptococcus salivarius in various solvents

The intrinsic viscosities, weight-average molecular weights (M?_w), and radii of gyration [($\text{R}{}^2{}_{\mathrm{g}}$)^{$\text{1}\text{2}$}≈] of Streptococcus salivarius levan in various solvents were respectively obtained from viscosity and light-scattering measurements. The data showed that the levan in water is not aggregated by hydrogen bonds, and that the values of both the refractive index and ($\text{R}{}^2{}_{\mathrm{g}}$)^{$\text{1}\text{2}$} are lower in water than in aqueous solutions of urea. Urea may break intramolecular hydrogen-bonds, e.g., between branches, allowing the molecule to expand.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

pH-Abhängigkeit der 13C-15N-kopplungskonstanten 15N-hochmarkierter aminosäuren,gewonnen aus algenmassenkulturenpH dependence of 13C-15N coupling constants of highly 15N-enriched amino acid isolated from mass cultivation of algae

The alga Ankistrodesmus braunii was grown with [¹⁵N]nitrate under the optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The ¹⁵N-labelled amino acids (¹⁵N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The ¹⁵N content of the analytically pure amino acids was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the ¹⁵N analysator. Using pulse Fourier transform ¹³C nuclear magnetic resonance, the pH dependence of the ¹³C-¹⁵N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J. Am. Chem. Soc. 86, 5564–5570) between the coupling constant and the product of the S-part of the ¹³C and ¹⁵N hybridization $\text{S}{}_{\mathrm{C}}\text{·}\text{S}{}_{\mathrm{N}}\text{= 80 · J (}{}^{13}\text{C}\text{-}{}^{15}\text{X}\text{)}$ fits best in acidic medium. The magnitude of the coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153–182). The recording of ¹³C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the ¹³C-¹⁵N coupling constants of the amino acids.     

Characterization of sodium and proton flows in sub-bacterial particles of Halobacterium halobium in terms of nonequilibrium thermodynamics

A thermodynamic characterization of the Na⁺-H⁺ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na⁺-H⁺ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, $\text{L}{}_{\mathrm{<mtext>H</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H\; +</mtext>}}\text{?Δ}\text{pH}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pNa</mtext>}}$, was in the range of 850–1150 nmol H⁺·(mg protein)^?1·h^?1·(pH unit)^?1 for four different preparations; for the total Na⁺ permeability, $\text{L}{}_{\mathrm{<mtext>Na</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 1620–2500 nmol Na⁺·(mg protein)^?1·h^?1·(pNa unit)^?1; and for the proton ‘cross-permeability’, $\text{L}{}_{\mathrm{<mtext>HNa</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 220–580 nmol H⁺·(mg protein)^?1·h^?1·(pNa unit)^?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na⁺-H⁺ exchange (η_max), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; η_max ? 5%; 0.36 ? r ? 2; ?_JNa⁺ ? 1.3 · 10⁵μmol · (RT unit)^?1 at J_Na = 1 μmolNa⁺ · (mgprotein)^?1 · h^?1; and ?_ΔpNa ? 5 · 10⁴ ΔpNa · (mg protein) · h · (RT unit)^?1 at ΔpNa = 1 unit, for different preparations.     

Proton transfer in and polarizability of hydrogen bonds coupled with conformational changes in proteins. II. IR investigation of polyhistidine with various carboxylic acids

Polyhistidine-carboxylic acid systems are studied by ir spectroscopy. It is shown that OH ?N ? O^?…H⁺N bonds formed between carboxylic groups and histidine residues are easily polarizable proton-transfer hydrogen bonds when the pK_a of the protonated histidine residues is about 2.8 units larger than that of the carboxylic groups. From these results it bis concluded that OH ?N ? O^? ?H⁺N bonds between glutamic or aspartic acid histidine residues in proteins may be easily polarizable proton-transfer bonds. Furthermore, it is demonstrated that water molecules shift the proton-transfer equilibria in these hydrogen bonds in favor of the polar structure, i.e., due to water or polar environments OH ?N ? O^? ?H⁺N bonds with smaller ΔpK_a values become easily polarizable proton-transfer hydrogen bonds. A consideration of the amide bands of polyhistidine shows that it can be present in five different conformations. It is shown that these conformational changes are strongly related to the degree of proton transfer. Hence, the degree of proton transfer, the degree of hydration, and conformation are not independent of each other, but are strongly coupled. Further proof for the interdependence of proton transfer and conformational changes are hysteresis effects, which are observed with studies of polyhistidine dependent on carboxylic acid, adsorption and desorption. OH ?N ? O^? ?H⁺N bonds between aspartic and glutamic acid and histidine residues are present in hemoglobin, in ribonucleases, and in proteases, whereby this type of bond is preferentially found in the active centers of these enzymes. It is pointed out that hydrogen bonds with such interaction properties should be of great significance for structure and especially functions of proteins in which they are present.     

31P and 17O NMR of Mn(III)-containing acid phosphatase: Evidence for direct metal-phosphate interaction and oxygen exchange from water into inorganic phosphate

The acid phosphatase isolated from sweet potato tubers by us is unique Mn(III)-containing enzyme which hydrolyzes phosphomonoesters and nucleotide phosphates. The present ³¹P and ¹⁷O NMR studies of the Mn(III)-containing acid phosphatase solved two important problems. The broadening of the phosphate ³¹P resonance signal in the 1:1 enzyme-substrate system shows evidence for direct metal-phosphate interaction in the Mn(III)-containing acid phosphatase. In addition, the ¹⁷O NMR evidence for oxygen exchange from water into inorganic phosphate strongly indicates that the Mn(III)-containing acid phosphatase catalyzes an apparent transition state displacement and P-O cleavage as follows: $\text{ROPO}{}_3{}^{\mathrm{=}}\text{+ H}{}^{17}\text{OHROH + H}{}^{17}\text{OPO}{}_3{}^{\mathrm{=}}$.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

Inhibition of chymotrypsin by alkyl phosphonates: a quantitative structure-activity analysis.

A quantitative structure-activity relationship has been formulated for 53 alkyl phosphonates [R₂OPO(CH₃)SR₃] inhibiting chymotrypsin: . In this so-called bilinear model, k_i is the bimolecular rate constant (m^?1 s^?1), β is a disposable parameter evaluated by a computerized iterative procedure, MR is the molar refractivity of a substituent, $\text{σ}{}_3{}^1$ is Taft's polar parameter, and I is an indicator variable for substituents containing a sulfonium group. The correlation coefficient for this equation is 0.985. This quantitative structure-activity relationship is compared with those previously formulated for the action of chymotrypsin on acylamino acid ester substrates.     

Structural and functional studies of Hemoglobin Wayne: An elongated α-chain variant

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated α-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.60}$ and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect ($\text{?Δ}\text{log}\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{/}\text{pH}\text{(7}\text{to}\text{8) = 0.34}$ and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l′ = 5.6 × 10⁶m^?1s^?1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l′ = 2.6 × 10⁶m^?1s^?1), whereas Hb Wayne Asp showed heterogeneous binding (l′ = 6.1 × 10⁶ and 2.6 × 10⁶m^?1s^?1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.     

Structural study of the solutions of acidic polysaccharides. I. Study of the solutions of acidic polysaccharides by measuring the activity coefficients of counterions

The activity coefficient of sodium ions in the presence of acidic polysaccharides (alginates, pectins and κ-carrageenan) has been measured potentiometrically. The activity determined in limit-dilute solutions normally exceeded the values calculated from the Manning theory. The difference (ΔγNa+) was regarded as a measure of the chain flexibility. The change in ΔγNa+ when a polysaccharide is transferred from water to 8 m urea solution gave information about the contribution of hydrogen bonds to the stabilisation of the polysaccharide conformation in aqueous solutions. The function n is independent of polymer concentration at high concentration. where γNa+exp is the experimentally determined value of the activity coefficient of the counterions and γNa+NaCl is the activity coefficient of sodium ions in a NaCl solution of the same equivalent concentration as the polymer solution.This suggests that the polysaccharide solution has a microheterogeneous structure. The results of the viscosity measurements suggest that there is agreement between equilibrium thermodynamic and rheological evaluation criteria for the structure of polymer solutions.     

Amiloride stimulation of sodium transport in the presence of calcium and a divalent cation chelator

Amiloride in nM to μM concentrations stimulates the short circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) of the toad urinary bladder by as much as 120% when applied in conjunction with apical Ca²⁺ and a divalent cation chelator. A significant decrease in transepithelial resistance ($\text{R}{}_{\mathrm{<mtext>t</mtext>}}$) is observed simultaneously. This response is spontaneously reversible and its amplitude is dependent upon apical sodium concentrations. The stimulated $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ persisted when acetazolamide (1 mM) was introduced, HPO₄^2? substituted for HCO₃^? or SO₄^2? replaced Cl^?. Consequently, the increase in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is not due to the change of Cl^?, H⁺ or HCO₃^? flux. This behavior in a ‘tight’ epithelium may be related to the mechanism controlling apical sodium permeability.     

Reactions of the antitumor agent carminic acid and derivatives with DNA

The antitumor agent carminic acid 1a does not bind to DNA but nicks it slowly, more rapidly when reduced in situ, and still more rapidly when prereduced at the quinone moiety. The nicking process requires oxygen and is selectively inhibited by (i) superoxide dismutase, (ii) catalase, and (iii) free radical scavengers indicating the involvement of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{, H}{}_2\text{O}{}_2$, and OH^., respectively. The intermediacy of OH^. was supported by spin trapping with N-t-butyl-α-phenylnitrone and epr of the radical produced via the carminic acid semiquinone. The single strand scission of DNA by carminic acid requires two adjacent hydroquinone moieties in the chromophore since reduced methyl tetra-O-methylcarminate 1b is without effect although it binds weakly to DNA. Polarographic redox potentials for the reversible (2e, 2H⁺) reduction of 1a and 1b are ?0.736 ± 0.003 V and ?0.56 ± 0.010 V against SCE, respectively. The fact that daunorubicin and adriamycin produce more extensive DNA strand scission than carminic acid under comparable conditions of prereduction and on a molar basis is largely attributed to the assistance of intercalative binding afforded in the case of the anthracyclines.     

The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain

1. Formate inhibits cytochrome $\text{c}$ oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome $\text{aa}{}_3$. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome $\text{aa}{}_3\text{(a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}$) and in the half-reduced species ($\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}$). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the $\text{aa}{}_3$ spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of $\text{a}{}_3{}^{\mathrm{2+}}$ minus $\text{a}{}_3{}^{\mathrm{3+}}$, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome $\text{a}{}_3{}^{\mathrm{3+}}$) and azide (which induces peak shifts of cytochrome $\text{a}{}^{\mathrm{2+}}$ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome $\text{a}{}^{\mathrm{2+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$ is faster than its rate of dissociation from $\text{a}{}^{\mathrm{3+}}\text{a}{}_3{}^{\mathrm{3+}}\text{-}\text{HCOOH}$, especially in the presence of cytochrome $\text{c}$. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome $\text{c}$ reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]².5. Formate inhibition of ascorbate plus $\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenyl-enediamine}$ oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome $\text{c}$ reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome $\text{aa}{}_3$ level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.     

Regulatory interaction between calmodulin and ATP on the red cell Ca2+ pump

The interactions between calmodulin, ATP and Ca²⁺ on the red cell Ca²⁺ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ by 5–10-fold and the rate of Ca²⁺-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ along a biphasic concentration curve ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>1</mtext>}}\text{≈ 1.4 μ}\text{M}\text{, K}{}_{\mathrm{<mtext>m</mtext><mtext>2</mtext>}}\text{≈ 330 μ}\text{M}$), but in stripped membranes the curve is essentially hyperbolic ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{≈ 7 μ}\text{M}$). In calmodulin-bound membranes Ca²⁺ activates $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ at low concentrations ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{< 0.28 μ}\text{M}$) in stripped membranes the apparent Ca²⁺ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E₂ and E₁ forms of the Ca²⁺ pump protein.     

Sulphate transport by rat ileum. Effect of molybdate and other anions

Kinetic constants for SO₄^2? transport by upper and lower rat ileum in vitro have been determined by computer fitting of rate vs concentration data obtained using the everted sac technique. MoO₄^2? inhibition of this transport is competitive, and kinetic constants for the inhibition were similarly determined. Transport is also inhibited by the anions WO₄^2?, S₂O₃^2? and SeO₄^2?, in the order $\text{S}{}_2\text{O}{}_3{}^{\mathrm{2?}}\text{> SeO}{}_4{}^{\mathrm{2?}}\text{≧ MoO}{}_4{}^{\mathrm{2?}}\text{> WO}{}_4{}^{\mathrm{2?}}$. These anions have no effect on the transport of l-valine. Low SO₄^2? transport rates were observed in sacs from animals fed a high-molybdenum diet. The significance of the results with respect to the problem of molybdate toxicity in animals is discussed, and related to the known protective effect of SO₄^2?.