Light sensitive zinc content of protein fractions from bovine rod outer segments

Bovine retinas, isolated rod outer segments and emulphogene extracts of rod outer segments have been shown to contain appreciable amounts of Zn²⁺, Ca²⁺ and Mg²⁺ when isolated in the absence of added metal ions. Chromatography of emulphogene extracted rod outer segments in Sephadex G-25 showed virtually all the Ca²⁺, Zn²⁺ and protein to elute with the void volume. Levels of Zn²⁺ but not Ca²⁺ were light sensitive. The Zn²⁺ contents of protein fractions were about 60% higher when samples were bleached. Under optimal conditions protein fractions contained 1.4 – 1.8 g atoms Zn²⁺/mole rhodopsin for dark adapted samples and 2.1 to 3.2 g atoms Zn²⁺/mole of rhodopsin for bleached samples.     

Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg.

Parthenogenetic activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. Parthenogenetic treatments, including the nonelectrolyte urea, hypertonic sea water, and ionophore A23187, all acted to release Ca²⁺ from intracellular stores. Ionophore and urea solutions release Ca²⁺ from the same intracellular store as normal fertilization. This intracellular store can be reloaded after 40 min and discharged again. Hypertonic medium appears to release Ca²⁺ from a different intracellular store. Treatment with the weak base NH₄Cl did not release intracellular Ca²⁺ but did result in a momentary Ca²⁺ influx if Ca²⁺ was present in the external solution. Ca²⁺ influx was not required for ammonia activation.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Kinetics and mechanism of oxidation of d-erythrose and dL-glyceraldehyde by chromium(VI) and vanadium(V) in perchloric acid medium

The kinetics of oxidation of d-erythrose and dL-glyceraldehyde by chromium (VI) and vanadium(V) in perchloric acid medium have been investigated spectrophotometrically. Each reaction was first-order with respect to [oxidant] and [substrate]. The reactions were catalysed by acid, but their dependence on acidity was complex. Sodium perchlorate accelerated the rate of each reaction. The oxidation rates follow the order glyceraldehyde > erythrose. The activation parameters were calculated and mechanisms consistent with the experimental observations are proposed.     

Evidence for an intermediate in the denaturation and assembly of phosphoglucose isomerase

The denaturation of dimeric rabbit muscle phosphoglucose isomerase in guanidine hydrochloride occurs in two discrete steps consisting of partial unfolding followed by subunit dissociation. In 3.5 to 4.5 m guanidine hydrochloride the enzyme forms a stable denaturation intermediate. Formation of this intermediate abolishes catalytic activity, shifts the protein fluorescence emission maximum from 332 to 345 nm, exposes all of the unavailable sulfhydryl groups, and decreases the s_20,w from 6.8 to 4.6 S. The intermediate dissociates into fully unfolded polypeptide chains with further increases in the concentration of the denaturant. The fluorescence maximum shifts to 352 nm and the s_20,w of the denatured monomer is 1.6 S. From the equilibrium constant for subunit association, 3 × 10⁴M^?1, in 4.7 m guanidine hydrochloride, the apparent free energy of association is estimated to be ?6 kcal mol^?1. Reconstitution of the enzyme protein takes place by the reversal of the steps observed upon denaturation. The denatured monomers refold and associate to reform the dimeric intermediate which then anneals to yield the intact enzyme molecule.     

A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 m sodium perchlorate is used for the final purification step.     

Interleukin 2 regulation of mitogen induction of immune interferon (IFN gamma) in spleen cells and thymocytes

Interleukin 2 (IL 2) or T-cell growth factor induced the production of immune interferon (IFN_γ) in C57B1/6 mouse spleen cell cultures, and enhanced mitogen-induced IFN_γ production in both spleen cells and thymocytes. Staphylococcal enterotoxin A, but not phytohemagglutinin P (PHA-P), induced IFN_γ production in thymocytes. IL 2 enhanced this production by almost 12-fold, while having no effect on the negative response to PHA-P. The IFN activity was shown to be IFN_γ by neutralization with specific antiserum. A strict correlation between IL 2 induction or enhancement of IFN_γ production and cell proliferation was not observed, probably indicating that non-IFN-producing cells also proliferated in the presence of IL 2. The data indicate that IL 2 can both induce IFN_γ and modulate mitogen induction of IFN_γ.     

Tomato alcohol dehydrogenase: Purification and substrate specificity

Alcohol dehydrogenase of tomato (Lycopersicon esculentum) has been purified to homogeneity, using affinity chromatography on Cibacron F3GA-agarose. The enzyme is a dimer, M_r 90,000–100,000. The coenzyme is NAD⁺; no NADP⁺-dependent activity was detected even in crude extracts. Among saturated substrates, ethanol and acetaldehyde show the lowest apparent K_m values (2.67 and 0.174 mm, respectively) and highest V values, supporting a primary role in acetaldehyde metabolism, with action also on “flavor aldehydes”; 2-unsaturated alcohols show still lower K_m values, probably due to a more favorable K_eq. This enzyme and other plant alcohol dehydrogenases form a definite class, intermediate in specificity between liver and yeast alcohol dehydrogenases: they differ from the former in being essentially inactive on secondary and aromatic substrates, from the latter in showing only a mild decrease in V with increasing chain length of alkyl substrates, and from both in showing the lowest K_m as well as highest V on ethanol and acetaldehyde. The tomato enzyme differs from other reported plant enzymes in showing substantial activity on geraniol. Kinetic studies are in agreement with an ordered sequential mechanism. The enzyme is inhibited slowly by iodoacetamide, and reversibly by acetamide and zinc-chelating compounds.     

The inhibition of L-3-hydroxyacyl-CoA dehydrogenase by acetoacetyl-CoA and the possible effect of this inhibitor on fatty acid oxidation

Acetoacetyl-CoA was found to strongly inhibit the dehydrogenation of L-3-hydroxybutyryl-CoA catalyzed by L-3-hydroxyacyl-CoA dehydrogenase from pig heart. The inhibition constant (K_i) was determined to be 7.7 × 10^?6 M, a value which is similar to the K_m value of 12 × 10^?6 M obtained for acetoacetyl-CoA in its NADH-dependent reduction catalyzed by the same enzyme. A suggested ordered BiBi mechanism for this enzyme, with NAD binding to the enzyme first, explains the observed noncompetitive nature of this inhibition. The possible effect of this inhibition on fatty acid oxidation is discussed.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

Synthesis of phenyl 2-acetamido-2-deoxy-4-O-α-l-fucopyranosyl-β-d-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-6-O-α-l-fucopyranosyl-β-d-glucopyranoside

Maize and potato amylopectin (57 and 64%, respectively) were recovered as non-cyclic products from 4-h digests of the starches with cyclodextrin glycosyltransferase {(1→4)-α-d-glucan:[(1→4)-α-d-glucopyranosyl]transferase (cyclising), EC 2.4.1.19} from Klebsiella pneumoniae M 5 al. Besides smaller saccharides, highly branched fragments of different sizes (average d.p. 40–140) were obtained by fractionation. The extents of beta-amylolysis varied between 24 and 37%, indicating that the clusters were not equally susceptible to attack by cyclodextrin glycosyltransferase. The fragments of potato amylopectin still contained larger amounts of material of high molecular weight. Accordingly, part of the longer B-chains of the basic structure were protected from the enzymic attack, presumably because of interchain branches. By debranching with pullulanase, it was evident that the beta-limit dextrins of the fragments of potato amylopectin were composed of longer B-chains (average chainlength 17.8) than those of maize amylopectin (average chain-length 14.1). The A/B-chain ratios, which were calculated from h.p.l.c. data for the debranched beta-limit dextrins, were 1.22 (maize) and 1.06 (potato). Some structural differences between potato and maize amylopectin are discussed.     

The induction of at least two distinct types of interferon in mouse spleen cell cultures by Corynebacterium parvum

Mice immunized with particulate antigens or soluble antigens in Freund's complete adjuvant produce a factor(s) which enhances antibody formation. Such an enhancing factor(s) is detected in the serum within 6 hr after immunization. The factor(s) is specific and enhances both 19s and 7s responses especially when recipient mice are challenged with subimmunogenic doses of antigen. From the study of the kinetics of antibody formation (latent period, coincidence of peaks of 19s and 7s responses) and the distribution of the Ig classes of the antibody (dominance of IgG1) it is concluded that the enhancing factor(s) primes the animals for a secondary response. The enhancing factor(s) is carrier specific and enhances antibody formation in the absence of T cells (nude mice). In the absence of T cells the antibody response is quantitatively small, which suggests that in the presence of T cells the enhancing factor(s) further amplifies antibody formation.     

Specific adsorption of starch oligosaccharides in the gel phase of starch granules

The gel phase of native starch-granules is penetrable by such low-molecular-weight solutes as oligosaccharides, amino acids, and salts [Lathe and Ruthven, Biochem. J., 62 (1956) 665]. Molecules larger than about 1000 daltons are effectively excluded. Starch oligosaccharides (maltotriose through maltoheptaose and perhaps higher) exhibit anomalous behavior in that they are taken up by the gel phase far in excess of the amount expected on the basis of their molecular size. Adsorption was measured by using radioactive starch oligosaccharides and counting weighed amounts of solution before and after equilibration with starch granules. The measurements were corrected for water sorption by the starch granules and for exclusion effects as ascertained by controls with nonstarch types of oligosaccharides. Maximum adsorption was observed with maltotetraose. The results indicate a specific binding between the starch oligosaccharides and molecular chains in the starch, presumably those chains in the gel phase. We suggest that these chains constitute interbranch regions of branched molecules, or segments of linear molecules in the gel or amorphous phase, the segments being of sufficient length to form a double helix or other association with the linear oligosaccharides.     

Reversible covalent modification of DNA

The reaction of bromomethylbenzoyl esters of choline and dimethylaminoethanol with DNA and model compounds led predominantly to phosphotriester formation. In model compounds the phosphotriester formation was verified by uv spectrometry. The bromomethylbenzoyl cationic esters reacted with DNA at room temperature at neutral pH values. The amount of the reagent chromophores was assessed semiquantitatively by spectrophotometry. The maximum binding appeared to be stoichiometric, i.e., one residue per phosphorus. The binding of one mole of reagent per phosphorus was confirmed by electron spectroscopic measurements of the phosphorus atom electron emission of maximally modified DNA. The modified DNA showed altered CD spectra indicating that the reagent chromophores are arranged in an orderly fashion affording a strong (Δ_? > 4), positive, apparently extrinsic CD band at ~240 nm; a double helical array is proposed. The introduced chromophores were readily removed by heat treatment or by treatment with nucleophiles at neutral pH values at moderate temperatures (<37 °C); no measurable fraction of the DNA became dialyzable. A decrease in viscosity accompanied the reversal, indicative of some chain breaking. The modified DNA's show higher T_m values than the native DNA and some display a higher and some a lower degree of cooperativity in their melting curves. No chemically detectable amounts of base alkylation, depurination, or depyrimidination were found when dialyzates of treated DNA and hydrolyzed samples of modified DNA were examined. However, presumptive evidence for some base alkylation by these novel alkylating agents was found utilizing Salmonella typhimurium tester strains sensitive to reversion by alkylation. No comparable binding of benzoylcholine, a nonalkylating analogue, by DNA was seen under conditions utilized here.     

Specific nucleotide sequences in HeLa cell inverted repeated DNA: enrichment for sequences found in double-stranded regions of heterogeneous nuclear RNA

Inverted repeat DNA was isolated from HeLa cell nuclei and transcribed in vitro with Escherichia coli RNA polymerase in the presence of [alpha-³²P]nucleoside triphosphates. The RNA products were digested with T₁ ribonuclease and subjected to separation in two dimensions. The pattern of the prominent oligonucleotides was almost indistinguishable from that seen when the double-stranded regions from ³²P-labeled HeLa cell heterogeneous nuclear RNA were fingerprinted in a similar manner. The sequences of several of the largest prominent T₁ ribonuclease-generated oligonucleotides were determined and were found to agree with those isolated from the double-stranded heterogeneous nuclear RNA that migrated to the same positions in the fingerprints. The most prominent component of the inverted repeat DNA appears to be sequences that are transcribed into double-stranded regions in heterogeneous nuclear RNA molecules.     

The effect of 20-hydroxyecdysone on synthesis of chromosomal and cytosol proteins in imaginal discs

Over 600 cytosol and 300 nonhistone chromosomal proteins of mass-isolated imaginal discs of Drosophila melanogaster have been resolved by two-dimensional electrophoresis. More than half of the nonhistone chromosomal proteins fall into families with effectively constant apparent molecular weight but varying isoelectric points. At least six chromosomal proteins differ distinctly in proportions between embryos and imaginal discs. The synthesis of six cytosol proteins is increased, and one decreased with incubation of the discs in vitro with 20-hydroxyecdysone. Two disc acidic chromosomal proteins are specifically synthesized in the presence of 20-hydroxyecdysone. Their isoelectric points and molecular weights are similar to those of the subunits of vertebrate steroid hormone receptor proteins. However, although ecdysteroid receptor activity is associated with purified chromatin, no ecdysteroid-dependent increase in receptor activity is detected during in vitro culture of discs.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

Studies on the orientations of the mitochondrial redox carriers I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45° between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2–1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a₃. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90°.