Kinetic alpha-deuterium isotope effects for enzymatic and nonenzymatic hydrolysis of nicotinamide-beta-riboside.

The kinetic α-secondary deuterium isotope effect, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}$, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp² character at the Cl′ position in the transition state for this reaction. A similar result, $\text{k}{}_{\mathrm{H}}\text{k}{}_{\mathrm{D}}\text{= 1.15}$, was obtained for base-catalyzed hydrolysis of NAD⁺. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.     

Influence of sterols on ion transport through lipid bilayer membranes

Charge-pulse relaxation experiments with the negatively charged lipophilic ions, dipicrylamine and tetraphenylborate, (as well as with the positively charged carrier system Rb⁺-valinomycin) have been carried out in order to study the influence of sterols on the ion transport through the lipid bilayer membrane. The mol fraction of the sterols (cholesterol, epicholesterol, ergosterol, stigmasterol, dihydrocholsterol, epicoprostanol and cholesterololeate) as referred to total lipid was varied in a wide range (mol fractions 0–0.8).The monoolein/sterol or dioleoylphosphatidylcholine/sterol mixtures were dissolved in $\text{n}$-hexadecane in order to minimize effects of the sterol on the membrane thickness.Cholesterol had a strong influence on the transport of the lipophilic ions. Its incorporation into monoolein membranes increased the rate constant ${}_{\mathrm{<mtext>i</mtext>}}$ of translocation up to 8-fold, but incorporation into phosphatidylcholine membranes had virtually no influence on $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$. The other sterols with one hydroxy group and cholesterololeate had no influence on the rate constant or the partition coefficient β. The results are discussed on the basis of a possible change of dipole potential of the membrane caused by cholesterol and its derivatives.In the case of valinomycin-mediated Rb⁺ transport only cholesterol had a strong influence on transport properties. The rate constants of association ($\text{k}{}_{\mathrm{<mtext>R</mtext>}}$) as well as the rate constants of translocation of the complex ($\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$) and of the free carrier ($\text{k}{}_{\mathrm{<mtext>S</mtext>}}$) were reduced by incorporation of cholesterol up to eight-fold. The decrease of $\text{k}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{k}{}_{\mathrm{<mtext>MS</mtext>}}$ are possibly caused by a decrease of membrane fluidity, whereas the decrease of $\text{k}{}_{\mathrm{<mtext>R</mtext>}}$ may be due to an increase of surface potential. The different action of cholesterol on the two transport systems is discussed under the assumption that the adsorption plane of the lipophilic ion is located more towards the aqueous side and that of the ion-carrier complexes more towards the hydrocarbon side of the dipole layer.     

Catalysis of ester hydrolysis by N-(2-dimethylaminoethyl)acetohydroxamic acid (I)

N-(2-dimethylaminoethyl)acetohydroxamic acid was synthesized. This compound, which incorporates a dimethylamino group as a second functionality into the hydroxamic acid molecule, catalyzes the hydrolysis of p-nitrophenyl acetate faster than acetohydroxamic acid itself does. The function of the dimethylamino group is to labilize the intermediate formed in the reaction, thus assisting deacylation intramolecularly. The dimethylamino group carries out this function by intramolecular general base catalysis. Nucleophilic catalysis is ruled out by the sizable deuterium oxide solvent isotope effect () found. General acid-hydroxide ion catalysis is ruled out by determination of the lack of reaction with azide ion, which does not possess a dissociable proton, with the intermediate in this reaction. The deuterium oxide solvent isotope effect on the azide ion reaction of the intermediate also rules out a general acid-hydroxide ion reaction.     

Characterization of the β-adrenergic receptors of hamster white fat cells: Evidence against an important role for the α2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the β-adrenergic antagonist, [³H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±1.3}\text{nM}$) but low capacity ($\text{32±17}\text{fmol/mg}$ membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±0.5}\text{nM}$) binding sites with a binding capacity of $\text{53 ± 13}\text{fmol/mg}$ membrane protein (corresponding to $\text{4000 ± 980}$ sites per cell), and independent kinetic analysis provided a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a $\text{β}{}_1\text{-}\text{receptor}$ subtype, yielding $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values in good agreement with the $\text{K}{}_{\mathrm{<mtext>act</mtext>}}$ and the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [³H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the $\text{α}{}_2\text{-}\text{component}$ of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the $\text{α}{}_2\text{-}\text{receptors}$ in the adrenergic control of lipolysis in hamster white adipocytes.     

Influence of coupling conditions on activity and operational stability of glucoamylase immobilized on titanium (IV)-activated controlled pore glass

Glucoamylase (EC 3.2.1.3) was coupled to controlled pore glass by using titanium(IV) chloride. The drying conditions used during the activation step were studied, and the highest activity (237 units/g of matrix) of immobilized enzyme was obtained when the support and the titanium(IV) chloride solution were dried at 45°C in vacuo for 16 h. After several washing cycles, the specific activity of the immobilized enzyme was ～13 units/mg of protein irrespective of the washing cycle used. However, this immobilized enzyme preparation was also the least stable ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1}\text{h}$). Investigation of the possibility of the stabilization of the linkage of the enzyme to the support by crosslinking with bifunctional reagents showed that the stabilization of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=100}\text{h}$) was achievable by treatment with a 5% glutaraldehyde solution at pH 7.0 for 2 h (product activity 67 units/g of matrix, specific activity 4 units/mg of protein); this product also showed no release of protein during use. A higher activity (296 units/g of matrix was achieved by stabilization by treatment with a 5% tannic acid solution at pH 7.0 for 2 h. The combined use of glutaraldehyde and tannic acid was effective in stabilizing the bound enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=80 to 120 h}$) with an initial activity of 116 units/g of matrix. When use was made of the same support in presilanized (3-aminopropyltriethoxy silane) form followed by glutaraldehyde coupling a similar initial activity (112 units/g of matrix) was obtained, but the operational stability was much better ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 640}\text{h}$.     

Superoxide anion as a cofactor of dopamine-beta-hydrxylase.

Superoxide anion can serve a reducing agent for tyramine hydroxylation by dopamine-β-hydroxylase. Stable $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ solutions were obtained by dissolving KO₂ in dry dimethylsulfoxide and infused into buffered solutions of tyramine and dopamine-β-hydroxylase at constant rate. The reaction requires molecular oxygen, but differs from the ascorbate dependent hydroxylation in its alkaline pH optimum value (pH 7.5) and its low rate (9 nmol octopamine formed/min/mg of protein). In absence of tyramine $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ does not produce a stable reduced form of the enzyme.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

Reactivity of chemically generated superoxide radical anion with peroxides as determined by competition kinetics

A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion ($\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$) with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$. High pressure liquid chromatography is used as an assay system for $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Active site studies of human thrombin and bovine trypsin: peptide substrates.

Twenty-four oligopeptides modeled after the N-terminal portion of the α(A)-chain of human fibrinogen were synthesized and tested as substrates for human thrombin and bovine trypsin. The peptides contained either an Arg-Gly bond, or an Arg-Val bond, or both. Glycine and glutamic acid were substituted at various positions within the peptides, and from the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for each peptide with thrombin and with trypsin, the importance of residues on both sides of the Arg-Gly bond was evaluated. The trypsincatalyzed hydrolysis is faster than the thrombin-catalyzed hydrolysis of the peptides with glutamic acid at various distances N-terminal to the Arg-Gly bond. The ratio of the thrombic rate to the tryptic rate is fastest when glutamic acid is six residues away. Thrombin selectively cleaves the Arg-Gly bond in Ac-Val-Arg-Gly-Pro-Arg-Val-OH but cleaves both arginyl bonds rapidly in Ac-Val-Arg-Gly-Pro-Arg-Val-OMe. Trypsin selectively cleaves an Arg-Val bond in Ac-Arg-Gly-Pro-Arg-Val-OMe. The results are discussed in the light of thrombic cleavages of proteins. Most of these are seen to occur at highly polar sequences that frequently contain a proline residue.     

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.     

Opposite modulation by uncoupling and electron transport limitation of the Kmapp of ADP for photophosphorylation

The $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K⁺) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold $\text{decrease}$ in the $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K⁺ are employed, the rate of photophosphorylation is decreased but a several-fold $\text{increase}$ in the $\text{K}{}_{\mathrm{m}}{}^{\mathrm{(app)}}$ of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient.     

Effect of PGF2α and neostigmine on induction of parturition,farrowing interval and litter survival in swine

An investigation of the effect of PGF₂α induced parturition alone or in combination with neostigmine (NEO) on subsequent productivity was conducted in a total confinement swine production unit. Forty-five crossbred gilts and 106 sows were randomly assigned to one of six treatments: (1) $\text{0 mg PGF}{}_2\text{α}\text{0 mg NEO}$, (2) $\text{10 mg PGF}{}_2\text{α}\text{0 mg NEO}$, (3) $\text{0 mg PGF}{}_2\text{α}\text{5 mg NEO}$, (4) $\text{10 mg PGF}{}_2\text{α}\text{5 mg NEO}$, (5) $\text{0 mg PGF}{}_2\text{α}\text{10 mg NEO}$, (6) $\text{10 mg PGF}{}_2\text{α}\text{10 mg NEO}$. PGF₂α injections (IM) were given 2 days prior to the mean expected farrowing day for this farm (day 112). NEO injections (IM) were given after the fourth pig born in gilt litters and after the fifth pig born in sow litters. Females injected with 0 or 10 mg PGF₂α farrowed 71.7 ± 3.7 and 31.4 ± 3.4 hours after treatment, respectively (P<0.01). No other parameters measured were significantly affected by injection of PGF₂α. These data show that PGF₂α effectively induced parturition and did not adversely affect subsequent litter productivity. NEO was found to be ineffective in reducing total farrowing time (P>0.05).     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

Characterization and application of a radioimmunoassay for beta-endorphin using an antiserum with negligible cross-reactivity against beta-lipotropin

High avidity antisera against β-endorphin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}\text{)}$ were obtained in two of five rabbits immunized with unconjugated synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{)}$ and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{61–65)}$ was 9%, while that with α-endorphin ($\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. Although this sequence is present in $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that $\text{α-N-}\text{acetyl}\text{-β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was not detectable (< 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/l. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was detectable ($\text{5 ± 3}\text{pmol/l}$; mean ± S.D.) after metyrapone. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. The specificity of the antiserum K-7762 was such that the $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.     

The effects of oxygen on alkaline solutions of superoxide dismutase.

The blowing of oxygen through strongly alkaline solutions of SOD leads to the drop of pH by more than 3 units. The rate of the process depends linearly on the concentration of SOD. The effect of oxygen on the modification of the shape and the decrease of the intensity of EPR signal of SOD were observed. The incubation of strongly alkaline solutions of SOD under vacuum leads to the reduction of the protein copper. The data obtained suggest, that the reduced copper may be at least partially reoxidized by oxygen. It is suggested that at pH 12.5 and higher in the presence of SOD the reaction of electron transfer from hydroxyl anion to the oxygen takes place: $\text{OH}{}^{\mathrm{?}}\text{+ O}{}_2\text{→ OH + O}{}^{\mathrm{?}}{}_{\mathrm{<mtext>2</mtext><mtext>?</mtext>}}$     

Solvent and substrate deuterium isotope effects on a transamination reaction catalyzed by pig heart aspartate aminotransferase.

Transamination of erythro-β-hydroxy-l-aspartate catalyzed by pig heart aspartate aminotransferase (EC 2.6.1.1) was studied with both normal and α-deuterated substrate in $\text{H}{}_2\text{O}\text{D}{}_2\text{O}$. The overall transamination reaction, with α-ketoglutarate as amino group acceptor, showed no primary substrate isotope effect. However, one of the elementary reactions between two enzyme-substrate complexes was found to exhibit large primary isotope effects in both the forward and the reverse directions. This same reaction also showed a twofold solvent isotope effect in the reverse direction, but D₂O had only a negligible effect in the forward direction. These data were interpreted to indicate that the substrate α-hydrogen arises from a Bronsted acid with two equivalent hydrogens. Another elementary reaction, which is 100-fold slower, was also studied since it appeared to be one of the principal rate-determining steps in the overall reaction. This step was not affected by substrate deuteration but exhibited large solvent isotope effects in both directions.     

Nitro analogs of substrates for adenylosuccinate synthetase and adenylosuccinate lyase

The reactivities of the nitro analogs of the substrates of adenylosuccinate synthetase and adenylosuccinate lyase, the enzymes which catalyze the penultimate and last step, respectively, in the pathway for AMP biosynthesis have been examined. Alanine-3-nitronate, an aspartate analog, was a substrate for the synthetase from Azotobacter vinelandii, having a $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ which was ~30% that for aspartate. The product of this reaction was N⁶-(l-1-carboxy-2-nitroethyl)-AMP. Of nine other substrate analogs tested, only cysteine sulfinate (having 5.5% of the activity of aspartate) was reactive. These results demonstrate the strict requirement of the synthetase for a negatively charged substituent, with a carboxylate-like geometry, at the β-carbon of the α-amino acid substrate. The lyase, purified to homogeneity from brewer's yeast by a new procedure, did not utilize N⁶-(l-1-carboxy-2-nitroethyl)-AMP as a substrate. However, the nitronate form of this analog was a good inhibitor of the lyase ($\text{K}{}_{\mathrm{m}}\text{K}{}_{\mathrm{i}}\text{= 28}$ when compared to adenylosuccinate), suggesting that it mimics a carbanionic intermediate in the reaction pathway. The avid binding of bromphenol blue by the lyase (_i = 0.95 μM) was used for active site titrations and for displacement of the enzyme, in the purification protocol, from blue Sepharose.     

The effect of ammonium ions upon isolated reactions of the glycolytic scheme

In the glycolytic system derived from rat brain acetone powder, ammonium ion has been found to stimulate three different reactions: (a) the transphosphorylase reaction from phosphoenolpyruvate, (b) the phosphohexokinase reaction, and (c) the hexokinase reaction. The transphosphorylases are affected differently depending upon whether adenosine diphosphate or adenylic acid is the phosphate acceptor; in the case of the latter, the dependency upon $\text{NH}{}_4{}^{\mathrm{+}}$ is particularly marked. A highly active myokinase is present in these extracts and its activity influences the transphosphorylase reaction to a considerable extent. The phosphohexokinase reaction is stimulated to a greater extent by $\text{NH}{}_4{}^{\mathrm{+}}$ than is the hexokinase reaction. In contrast to these reactions which require the participation of the adenylic system, triose phosphate oxidase activity is uninfluenced by the presence of $\text{NH}{}_4{}^{\mathrm{+}}$.