Synthesis of coenzymically active soluble and insoluble macromolecularized NAD+ derivatives.

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an omega-carboxyalkyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.     

Design of a new water-soluble pressure-sensitive adhesive for patch preparation

This work was intended to improve the adhesion properties of an available medical water-soluble pressure-sensitive adhesive (PSA) through the addition of cellulose ethers or polyvinylpyrrolidone (PVP). The adhesion properties were evaluated by means of peel adhesion test and creep resistance test. Possible interactions between the polymethyl methacrylate (PMMA) and hydrocolloid were investigated by Fourier-transformed infrared spectroscopy. Moreover, a central composite design was used to estimate the effects of hydrocolloids and plasticizers and their interactions on the PSA performance. The addition of PVP made it possible to obtain a patch with a 40-fold improved creep compliance and a reduced peel adhesion. The significant increase of the matrix cohesion was due to attractive interactions between the amide group of PVP and the carboxylic acid group of PMMA. The water vapor permeability of the prepared systems was very high. Furthermore, no primary skin irritation was observed. The presence of plasticizers at high level increased both the peel values and creep compliance, showing an opposite behavior with respect to PVP. The new PSA system can be easily removed from the skin, is suitable for repeated applications on the same site, and has adhesive properties that can be modified by changing the component ratios.     

Isolation from yeast of a metabolically active water-soluble form of ergosterol

A water-soluble complex containing ergosterol together with a component of yeast has been isolated. The complex can be isolated from commercial yeast extract to which ergosterol has been added or directly from whole yeast cells. The complexing component has the properties of a large polysaccharide and the binding between the sterol and the polysaccharide appears to be noncovalent. The complex is easily prepared and is stable in aqueous solution; ergosterol in this solution is metabolically available to yeast cells to which it is added.     

Enzymic preparation of water-soluble chitosan and their antitumor activity

Water-soluble low-molecular-weight (LMW) chitosan was prepared from enzymatic hydrolysis with efficient hemicellulase. The hydrolysates were separated by ultrafiltration membranes. A separated fraction with Mw more than 5x10(3) and with a degree of deacetylation of 58% was water-soluble in the free amine form. The intraperitoneal injection of LMW chitosan and its N-acetyl product inhibited the growth of sacroma 180 (S180) tumor cells in the mice, and the maximum inhibitory rate reached 64.2%. The oral administration was also effective on decreasing weight of tumor, and the maximum inhibitory rate reached 33.7%. The Water-soluble chitosan with higher Mw than hexamer might have better antitumor activity.     

Isolation of a highly active preparation of beta-D-galactosidase

Methods for isolation and purification of beta-galactosidase from Bacillus subtilis, st. IBP-101 are described. The bacterial cells were disrupted by different procedures such as freezing and thawing with subsequent autolysis at 37 degrees C, disrupting in a French press DKM-3 or in ultrasonic disintegrators UZDN-1 (USSR) and Soniprep-150. It is shown that the specific activity and yield of the enzyme depends to a great extent on the disrupting procedure used. The best results were obtained in case of sonication. The preparation was purified by precipitation with ammonium sulphate (25-75% saturated) and chromatography on DEAE-cellulose and DEAE-Sephadex. The purified enzyme had a specific activity of 3155 units per mg protein. The molecular weight of the homogeneous according to gel polyacrylamide electrophoresis preparation was 215,000, as estimated by gel filtration, and 105,000, as estimated by SDS gel electrophoresis. The enzyme retains the activity in the presence of Na+, Mn2+ or Mg2+ ions or the thiolic reagents, dithiothreitol or 2-mercaptoethanol. The pH optimum of the enzyme activity is 6.3 and it is stable in water solutions at pH from 6 to 9 and can be lyophilized. The given preparation of beta-galactosidase has a high affinity for synthetic substrates such as o- and p-nitrophenyl-beta-D-galactopyranosides and 4-methylumbelliferyl-beta-D-galactopyranoside.     

The preparation and characterisation of chromatin from third instar larvae of Drosophila melanogaster

A procedure is described for the isolation of large amounts of chromatin from Drosophila third instar larvae. This material is suitable for immunisation of rabbits, and preliminary analysis by immunodiffusion reveals little cross reaction between chromatin and cytoplasm antigens, using antisera prepared against them. In addition to these studies, results from an examination of the exchange of radioactively labelled cytoplasmic proteins with nuclei suggest that no more than 5% of the total chromatin protein is of cytoplasmic origin. Electrophoretic analysis indicates that of this 5%, 20% migrates as a single band, in the same position as f2b histone. The techniques of radioactively labelling larvae which were used, may be suitable for achieving specific activities of up to 100μC/mg larval protein. Contamination of the chromatin by yeast proteins from the larval food is no more than 10%. Of this 30% is represented by one electrophoretic band. Immunological analysis reveals little cross reaction with yeast extracts.     

Production of essential L-branched-chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values.     

The preparation and structural characterisation of thiolato anions of bismuth(III)

The preparation and structures of the bismuth thiolato anions [Bi₂(SC₆F₅)₆(μ-SC₆F₅)]⁻ and [Bi₂(SC₆F₅)₆(μ-SC₆F₅)₂]²⁻ and the halothiolato anions [Bi₂(SC₆F₅)₆(μ-Br)]⁻, [Bi₂(SC₆F₅)₆(μ-Cl)₂]²⁻ and [Bi₃(SC₆F₅)₉(μ-Br)₂]²⁻ are described. All compounds have been isolated from reactions between Bi(SC₆F₅)₃ and ammonium or phosphonium halides. The basic structural units in the dinuclear species are of two types namely [Bi₂(SR)₆(μ-X)]⁻ and [Bi₂(SR)₆(μ-X)₂]²⁻, where X=thiolate or halide. In the former case the single bridging groups occupy an axial site within the disphenoidal (equatorially vacant, trigonal bipyramidal) geometry around the bismuth centres whereas in the latter the two bridging groups occupy cis basal sites in square based pyramidal bismuth environments. The trinuclear anion [Bi₃(SC₆F₅)₉(μ-Br)₂]²⁻ has features in common with the basic [Bi₂(SC₆F₅)₆(μ-Br)]⁻ unit.     

Characterization of water-soluble products of palmitic acid beta-oxidation by a rat brain preparation

Abstract: (1) [1-¹⁴C]Palmitic acid was oxidized to CO₂ and a water-soluble material by a rat brain preparation. The radioactive CO₂ and water-soluble material were produced in a ratio of 1.0:1.3 when the mitochondrial fraction was used, and 1.0:10 or more with the postnuclear fraction. There was a lag period of 10 min for CO₂ production. These conversions were stimulated by carnitine and inhibited by cyanide. (2) Of the total radioactivity in the water-soluble material obtained with the mitochondrial fraction, 65% after 10 min of incubation and 80% thereafter were associated with amino acids, mostly with aspartate and glutamate. The remaining radioactivity, 35 and 20%, respectively, was associated with organic acids, 60–65% in citrate. The water-soluble material obtained with the postnuclear fraction contained an equal amount of radioactivity in organic and amino acids during the course of the experiment. In the organic acids, succinate was the highest labeled product during 10–40 min of incubation, whereas citrate was the highest labeled at the end of 60 min of incubation. After 60 min, the radioactivity in the amino acids was markedly associated with glutamate, and its radioactivity was 10 times greater with the postnuclear fraction than with the mitochondrial one. (3) An experiment with rat liver preparations was also camed out. The liver mitochondrial fraction showed an accumulation of radioactive organic acids within 10 min of incubation, which was followed by a linear production of ¹⁴CO₂. With the liver postnuclear fraction, the radioactivity was found mostly in the organic acids during the course of the experiment. In the liver system, the radioactive amino acids accounted for only 25% or less of the total radioactivity in the water-soluble material.     

The preparation and characterisation of native photoreceptor units from the thylakoids of Rhodopseudomonas viridis

The photosynthetic membranes of Rhodopseudomonas viridis consist of a regular array of structural units. Each unit is composed of a central core (thought to contain the reaction centre complex) surrounded by a subdivided ring of protein (of likely antennae function). These individual units can be dissociated from the membrances using a variety of detergent treatments. The absorption spectrum, used as a criterion of a native state, is retained. All of the seven major polypeptides, the four reaction centre polypeptides (cytochrome, H, M and L chain) as well as the three light-harvesting polypeptides (B1015-α, β and ξ) are shown to be present. Electron microscopy of the units shows a similar structure to the units within the membrane. surface-specific iodination of both membranes and units labels predominantly polypeptides H, B1015-α, and ξ. M and L are weakly labelled. In addition, B1015-β is labelled in the isolated units. This, with other evidence, supports an allocation of light-harvesting polypeptides to the outer ring. Further solubilisation of these units separates the reaction centre (as a native complex containing all four polypeptides) from the light-harvesting polypeptides. The light-harvesting polypeptides are obtained in a form containing all three polypeptides and bound pigment, however the peak at 1015 nm corresponding to native bacteriochlorophyll b is lost.     

The preparation and characterisation of some complexes of iron(II) with amino acids

The following complexes of iron(II) with the amino acids glycine, alanine, phenylglycine, phenylalanine, leucine, serine, aspartic acid, glutamic acid, glutamine, tryptophan, histidine, methionine, S-methylcysteine, cystine, and glycylglycine have been isolated: Fe(Gly)₂, Fe(Ala)₂, Fe(Phegly)₂, Fe(Phe)₂·2H₂0, Fe(Leu)₂·2H₂0, Fe(Ser)₂, Fe(Asp)·2H₂0, Fe(Glu)·2H₂0, Fe(Gln)₂, Fe(Trp)₂, Fe(His)₂·H₂0 and 2H₂0, Fe(Met)₂, Fe(MeCys)₂, Fe(CysCys) and Fe(GlyGly)₂. Their magnetic behaviour, reflectance spectra, and Mössbauer parameters are consistent with high spin, hexacoordinate iron(II), and imply extended structures involving carboxylate bridges.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg2+-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567-576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The Km value for ATP was 1 mM and apparent Km for Mg2+ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd2+ and Zn2+ salts, p-chloromercuribenzoate and N-ethylmaleimide, known inhibitors of membrane endocytosis.     

The preparation of fully active chymopapain free of contaminating proteinases

Chymopapain (EC 3.4.22.6) was purified from commercially available dried latex of papaya (Carica papaya) by extraction at acidic pH, cation-exchange chromatography and active site-directed affinity chromatography on immobilized alanyl-phenyl-alaninaldehyde semicarbazone, with elution by mercuric chloride. The product was found by immunoassay to be essentially free of the other cysteine proteinases from papaya, including papaya proteinase IV, and was fully active. The rate of alkylation of the active site cysteine of chymopapain by iodoacetate was found to be sufficiently rapid and selective for this reagent to be used as an active-site titrant.