Preparation and some physico-chemical properties of L-canaline

A new and simple method for the preparation of L-canaline for use in biological studies is described. This material, an analogue of ornithine, was prepared by enzymatic hydrolysis of L-canavanine followed by fractionation under physiological conditions by gel filtration on Sephadex G10. The behaviour in thin-layer chromatography of the separated material is identical with that of canaline prepared by two different routes of organic synthesis. Further physico-chemical characterisation gave data compatible with the molecular structure proposed for canaline (2-amino-4-aminoxybutyric acid). Addition of L-canaline results in a change in the absorption spectrum of pyridoxal phosphate, maximum effect being observed in the presence of equimolar amounts. Under these conditions, a single addition product is detected in thin-layer chromatography. Each of the canaline preparations tested is identical in this respect. The biological significance of this reaction is discussed.     

Anthracycline gels. Preparation and some physico-chemical properties

Gels have been prepared from aqueous solutions of anthracyclines by addition of salts. The gels are thixotropic and thermally reversible. They are stable for several months in the refrigerator and for long times even at room temperature. The gel-solution transition (melting) temperature depends on the concentration of the anthracycline and on the concentration and nature of the added salt. The melting has been followed by 1H-NMR. Only weak intermolecular interactions (stacking and hydrogen bonds) originate the drug network, within which the solvent is entrapped. 1H-NMR and polarimetric data suggest a stacked helical arrangement of the anthracycline molecules. The gelation process is cooperative.     

Ca2+-phospholipid-dependent protein kinase from the rat liver: physico-chemical properties and kinetic parameters

Ca2+, a phospholipid-dependent protein kinase, was isolated from the rat liver and the dependence of this enzyme on different conditions of the phosphotransferase reaction and substrate specificity was studied. The amino acid analysis was performed using "Aminochrom-2" (Hungary).     

Various physico-chemical properties of the Yersinia pestis capsular protein

Some properties of the structure of Y. pestis capsular antigen macromolecules have been studied. The aminoacid composition of F1 protein, the aminoacid sequence of the N-terminal fragment of antigen polipeptide chain were determined. Some peculiarities in the dissociation of capsular antigen macromolecules have been studied. The formation of the product resulting from unterminated thermodissociation of F1 protein oligomeric form, consisting of four subunits, has been registered. The aspects of F1 protein association are discussed.     

Purification and some physico-chemical properties of myocardial adenylate deaminase

A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.     

Antimicrobial activity and physico-chemical properties of some alkyldimethylbenzylammonium chlorides

A series of alkyldimethylbenzylammonium chlorides have been synthesized with n-alkyl chain lengths of C1 leads to C18. Octanol/water partition coefficients were determined and the antimicrobial activity assessed as the minimum growth inhibitory concentrations towards twelve strains of micro-organisms, representative of Gram-negative and Gram-positive bacteria, yeasts and fungi. The data were subjected to a numerical analysis. Antimicrobial activity of the compounds was found to be a parabolic function of their lipophilicity and maximized with n-alkyl chain lengths of between C12 and C16. The data fit to quadratic functions estimated for low (C1-C7) and high (C8-C16) alkyl chain length compounds was better than for a single quadratic describing the activity of the complete series (C1-C18). These maximized at log P values corresponding to alkyl-chain lengths of approximately C7 and C14 respectively, and were suggestive of low and high affinity binding sites upon the cell surface. The data analysis allowed the chain lengths of compounds with optimal activity towards the various groups of organisms to be determined. Generally yeasts and fungi were most sensitive towards C12, Gram-positive bacteria towards C14, and the Gram-negative bacteria towards C16. Gram-negative cells were the most resistant towards all the compounds and Gram-positive cells the least.     

Exploring strategies for protein trapping in Drosophila

The use of fluorescent protein tags has had a huge impact on cell biological studies in virtually every experimental system. Incorporation of coding sequence for fluorescent proteins such as green fluorescent protein (GFP) into genes at their endogenous chromosomal position is especially useful for generating GFP-fusion proteins that provide accurate cellular and subcellular expression data. We tested modifications of a transposon-based protein trap screening procedure in Drosophila to optimize the rate of recovering useful protein traps and their analysis. Transposons carrying the GFP-coding sequence flanked by splice acceptor and donor sequences were mobilized, and new insertions that resulted in production of GFP were captured using an automated embryo sorter. Individual stocks were established, GFP expression was analyzed during oogenesis, and insertion sites were determined by sequencing genomic DNA flanking the insertions. The resulting collection includes lines with protein traps in which GFP was spliced into mRNAs and embedded within endogenous proteins or enhancer traps in which GFP expression depended on splicing into transposon-derived RNA. We report a total of 335 genes associated with protein or enhancer traps and a web-accessible database for viewing molecular information and expression data for these genes.     

cGMP-dependent protein kinase genes in Drosophila

Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.     

Isolation and some properties of protein kinase from pigeon breast muscle]

Protein kinase was isolated from pigeon breast muscle. The preparation obtained was chromatographically homogeneous. The apparent Km varlue for histone H1 and ATP were 3,5-10(-5) M and 1,6-10(-5) M respectively. The purified enzyme displays high specificity for the lysine-rich histones (H1, H2b, H2a). The protein kinase activity is stimulated, 1,6-fold by cyclic AMP.     

Purification and some properties of Saccharomyces cerevisiae meiosis-specific protein kinase Ime2

Ime2 is the founding member of a family of protein kinases that are required for effective progression through meiotic development. Ime2 is essential for the induction of meiosis-specific genes and for the activation of meiotic DNA replication in the budding yeast Saccharomyces cerevisiae. Aside from the fact that Ime2 is a protein kinase and shares several amino acid motifs with cyclin dependent kinases, virtually nothing is known about its enzymatic properties or substrates. Biochemical characterization of Ime2 has been hindered by its low abundance and short half-life. We have created baculovirus expression vectors to produce recombinant Ime2 in insect cells. In this report, we describe the overproduction of Ime2 and its purification using affinity chromatography. Using this procedure, we have been able to purify up to 2mg Ime2 from 1L of infected insect cells. The Ime2 isolated by this method displays properties similar to those of the native enzyme that has been immunoprecipitated from yeast. The high level expression of Ime2 in this system and its ease of purification will be beneficial for more extensive biochemical analysis of Ime2 and related meiosis-specific kinases.