Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. II. Influence of pH on kinetics of electron transfer

pH Dependences of steady-state kinetic parameters of cytochrome chains of submitochondrial particles have been studied. It has been shown that the lifetimes of activated states (τ) of the pairs of cytochromes b → c₁ and a → a₃ have different pH dependences; those for the c₁ → c and c → a cytochrome pairs being similar. The rate constants for the non-activated state of the respiratory chains decreased for the b → c₁ pair and increased for the a → a₃ pair when the pH value was increased.The values of pK calculated from these dependences for the pairs b → c₁ and a → a₃ were 7.2 and 8.9, respectively. It has been supposed that the ratio of activated to non-activated electron carriers may be controlled by the local pH value in the mitochondrial membrane, the latter being dependent upon the rate of electron transfer. The kinetic model based on this assumption allows one to explain the experimental dependences on pH of the rate constants for cytochromes b → c, and a → a₃.The values of the diffusion rate constants for H⁺ and OH^? ions in the mitochondrial membrane estimated from these kinetic data obtained in this study weree 10⁴–10⁵ s^?1 and 10²–10³ s^?1, respectively.     

Purification and properties of myo-inositol-1-phosphate dehydrogenase from germinating mung bean seeds

A novel enzyme, myo-inositol-1-phosphate dehydrogenase, which catalyzes the conversion of myo-inositol 1-phosphate to ribulose 5-phosphate has been purified 84-fold from mung bean seedling employing several common techniques. The molecular weight of this purified enzyme has been recorded as 88,500 by Sephadex G-200 column chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis one protein band containing three subunits of M_r 32,000 each was discernible. K_m values for NAD⁺ and myo-inositol 1-phosphate have been recorded as 2.8 × 10^?4 and 5.0 × 10^?4m, respectively. Production of NADH in myo-inositol-1-phosphate dehydrogenase reaction has also been evidenced by measurement of NADH fluorescence. Dehydrogenation and decarboxylation of myo-inositol 1-phosphate are mediated by the same enzyme. In fact, the rate of dehydrogenation corroborates with that of decarboxylation. Stoichiometry of this reaction suggests that for the production of 1 mol of ribulose 5-phosphate 2 mol of NAD⁺ are reduced.     

Campestroside,a new tetrahydroxanthone glucoside from Gentiana campestris

Campestroside has been isolated from the aerial parts of Gentiana campestris. From UV, MS, ¹H-NMR and ¹³C-NMR data its structure has been established as 1,3,5-trihydroxy-8-β-d-glucopyranosyl-5,6,7,8-tetrahydroxanthone. Campestroside which has also been detected in G. ramosa and G. germanica is the first reported tetrahydroxanthone glycoside.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

Mukonal,a probable biogenetic intermediate of pyranocarbazole alkaloids from murraya koenigii

Mukonal, a carbazole alkaloid has been isolated from Murraya koenigii. The structure of the compound has been established as 2-hydroxy-3-formyl carbazole based on physical (UV, IR, ¹H NMR, ¹³C NMR and mass spectrometry) and chemical transformations.     

Argophyllone-B,a sesquiterpene lactone from Helianthus argophyllus

The sesquiterpene lactone, 2-methyl-2-butenoic acid dodecahydro-4-(hydroxymethyl)-10a-methyl-8-methylene-3,7-dioxooxineno[5,6]cyclodeca[1,2-b]furan-9-yl ester [1aR^*-[1aS^*,4R^*,5aS^*,8aR^*,9R^*(E)]], argophyllone-B, was isolated from acetone extracts from the leaves of Helianthus argophyllus. Its structure has been determined by single crystal X-ray analysis. Complete ¹H NMR and ¹³C NMR assignments have been made.     

Further characterization of phosphoinositol kinase isolated from germinating mung bean seeds

Phosphoinositol kinase isolated and purified from germinating mung bean seeds has been further characterized. The rate of phosphorylation varies with different inositol phosphates and this is consistent with the K_m and V_max for each of the substrates. The phosphate transfer from ATP has been found to be mediated by a phosphoprotein intermediate. In a particular step of the reaction the immediate product of the reaction has been found to be most inhibitory, other products being less or non-inhibitory. The inhibition has been found to be competitive in nature. The K_is have been found to range between 0.6 and 1 × 10^?4 M. ADP also inhibited non-competitively with respect to IP₅. K_i for this has been found to be 2.3 × 10^?4 M. The purified enzyme migrated as a single protein band on polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulphate it is dissociated into 3 subunits in the ratio 1 : 1 : 1. The MW of the three subunits are approx. 86 000, 56 000 and 35 000. The MW of the enzyme has been found to be approx. 177 000.     

(—)-Massoniresinol,a lignan from Pinus massoniana

A new lignan, named (—)-massoniresinol, has been isolated from Pinus massoniana needles. Its structure has been proved to be (2R,3S,4R)-3,4-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-(4-hydroxy-3-methoxybenzyl)-3-tetrahydrofuranmethanol by ¹H NMR, ¹³C NMR, mass and CD spectroscopy.     

Isolation of a cardiotonic glycoprotein,striatoxin, from the venom of the marine snail Conus striatus

Striatoxin, a powerful cardiotonic glycoprotein has been isolated from the venom of the marine snail Conus striatus, monitored by the inotropic action on the guinea-pig left atria. The molecular weight was estimated to be 25,000 by gel filtration. The purified glycoprotein is electrophoretically homogeneous. The toxin at concentrations above 10^?7 g/ml has a long-lasting inotropic action, which was abolished by tetrodotoxin (10^?6 M). The minimum lethal dose in the fish Rhodeus ocellatus smithi was 1 μg/g body weight. A possible biological role of striatoxin in C. striatus is briefly discussed.     

The reaction of 4,4'-bis-dimethylaminodiphenylcarbinol with the sulfhydryl group. A new reagent for sulfhydryl analysis

4,4′-bis-Dimethylaminodiphenylcarbinol (BDC-OH) dissociates in aqueous buffers at pH values below neutrality to form a resonance-stabilized carbonium-immonium ion (BDC⁺) which exhibits an absorbance maximum at 606 nm. In the presence of 4.0 M guanidine hydrochloride, BDC⁺ has an apparent molar absorption coefficient of 70,800 M^?1cm^?1 and an absorbance maximum of 612 nm. Sulfhydryl groups react with the cation to form S-(4,4′-bis-dimethylaminodiphenylmethyl-) derivatives with a concomitant quantitative loss of the 612-nm absorbance. This quantitative interaction has been exploited in the development of a new and convenient technique for the quantitative determination of sulfhydryl groups in proteins. Results of sulfhydryl determinations on simple thiols and five proteins are presented, along with comparison data obtained via other sulfhydryl techniques.     

Organo-peroxyl compounds via catalytic oxidation of a hindered phenol and aniline utilizing new manganese(II) bis benzimidazole diamide based complexes

Bis benzimidazole diamide ligand-N,N′-bis(2-methylbenzimidazolyl) propanediamide [GBMA = L] has been synthesized and utilized to prepare new Mn(II) complexes of general composition [Mn(L)X₂]·nH₂O where X is an exogenous anionic ligand(X = Cl⁻, CH₃COO⁻, SCN⁻). The geometry of the ligand and its Mn(II) complex have been optimized at the level of UHF, by using ZINDO/1 method. Binding energies, heat of formation and bond lengths of geometry optimized structures for the ligand and complex have been obtained. The oxidation of 2,4,6-tri-tert.-butylphenol (TTBP) and 2,4,6-tri-tert.-butylaniline (TTBA) has been investigated using these Mn(II) complexes as catalyst and TBHP as an alternate source of oxygen. The organo-peroxyl compounds have been isolated and characterized by ¹H NMR, ¹³C NMR, IR and mass data. A different product profile was obtained when H₂O₂ is used as an oxidant.     

The dissociation of insulin from human insulin antibodies

The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k₁ = 2·1^?3 min^?1 and a fast (k₂ = 4·10^?2 min^?1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k₁ and k₂ values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6–850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achivement of maximal binding there is a time-dependent rise in binding to the slow dissociating component, with a concomitant fall in k₁. The traditional concept that equilibrium is established at maximum binding requires further examination.     

Uvariastrol,a novel cycloartane triterpene from the stem bark of Uvariastrum zenkeri

Uvariastrol, a cycloartane triterpene with a novel tetrahydrofuran/furanone side-chain, has been isolated from the stem bark of Uvariastrum zenkeri and characterized using spectral characteristics, notably high-field ¹H NMR and ¹³C NMR spectroscopy.     

Metabolism of 14C-labelled D-glucose, D-fructose and allitol by Itea plants

The metabolism of D-[1-¹⁴C]glucose, D-[6-¹⁴C]glucose, D-[1-¹⁴C]fructose and D-[6-¹⁴C]fructose by leafy spurs of Itea plants results in rapid incorporation of label into allitol and D-allulose. The patterns of labelling found in the allitol and D-allulose are discussed, a direct interconversion from D-glucose and D-fructose being indicated. Allitol has been found to be an active metabolite in Itea plants.     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

5,7,3′-Trihydroxy-6,8-di-C-methyl-4′,5′-dimethoxyflavanone from Alluaudiopsis marnieriana

From bark and spines of Alluaudiopsis marnieriana, a novel flavanone has been isolated and identified as 5,7,3′-trihydroxy-6,8-di-C-methyl-4′,5′-dimethoxyflavanone by UV, ¹H NMR and mass spectroscopy.     

Wampetin,a furocoumarin from Clausena wampi

A new furocoumarin wampetin has been isolated from Clausena wampi (syn. Clausena lansium). The structure was established from ¹H NMR, ¹³C NMR, MS and chemical data.     

Amplification of ssb-1 mutant single-stranded DNA-binding protein in Escherichia coli

The ssb-1 gene encoding a mutant Escherichia coli single-stranded DNA-binding protein has been cloned into plasmid pACYC184. The amount of overproduction of the cloned ssb-1 gene is dependent upon its orientation in the plasmid. In the less efficient orientation, 25-fold more mutant protein is produced than in strains carrying only one (chromosomal) copy of the gene: the other orientation results in more than 60-fold overproduction of this protein. Analysis of the effects of overproduction of the ssb-1 encoded protein has shown that most of the deficiencies associated with the ssb-1 mutation when present in single gene copy, including temperature-sensitive conditional lethality and deficiencies in amplified synthesis of RecA protein and ultraviolet light-promoted induction of prophage λ⁺, are reversed by increased production of ssb-1 mutant protein. These results provide evidence in vivo that SSB protein plays an active role in recA-dependent processes. Homogenotization of a nearby genetic locus (uvrA) was identified in the cloning of the ssb-1 mutant gene. This observation has implications in the analysis of uvrA^? mutant strains and will provide a means of transferring ssb^? mutations from plasmids to the chromosome. On a broader scale, the observation may provide the basis of a general strategy to transfer mutations between plasmids and chromosomes.     

Hemoglobin-water interactions in normal and sickle erythrocytes by proton magnetic resonance T1ϱ measurements

The dependence of the water proton magnetic resonance spin-lattice relaxation rate (T_1?^?1) in the rotating frame on the strength of the spin-locking (H₁) field has been investigated for packed oxy and deoxy normal and sickle erythrocytes at temperatures from 9 to 40 °C. The T_1?^?1 of oxy or deoxy normal erythrocytes shows no dependence on H₁ up to ~7 G at any temperature studied. On the other hand, T_1?^?1 decreases from about 40 s^?1 to 15 s^?1 (H₁ from 0 to ~7 G) for deoxygenated packed sickle cells at 40 °C. The magnitude of this variation of T_1?^?1 with H₁ decreases with decreasing temperature. Oxy packed sickle cells also show a dependence of T_1?^?1 on H₁ but the magnitude is <10% of that of the deoxygenated samples. These results suggest that water proton T_1?^?1 measurements are a sensitive probe of hemoglobin S polymerization and provide a novel technique for the study of slow water motions in these systems. The T_1?^?1 results are compared with low frequency T₁^?1 results of other investigators on hemoglobin S solutions. Analysis of the data suggests that water proton motions with correlation times of the order of 10^?5 s are present in the deoxygenated sickle cell samples at temperatures above 10 °C.     

Structures of verbascoside and orobanchoside,caffeic acid sugar esters from Orobanche rapum-genistae

The complete structural elucidation of the two caffeic acid sugar esters verbascoside and orobanchoside, has been realized by ¹H and ¹³C NMR studies. It has been demonstrated that verbascoside is β-(3′,4′-dihydroxyphenyl)ethyl-O-α-L-rhamnopyranosyl(1→3)-β-D-(4-O-caffeoyl)-glucopyranoside, and orobanchoside is β-hydroxy-β-(3′,4′-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl(1→2)-β-D-(4-O-caffeoyl)-glucopyranoside.