N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploiting the cell membrane for the production of heterologous proteins in Escherichia coli

The bacterial membrane serves both as a cell organelle and as a barrier for segregating the metabolically active cytoplasm from the extracellular milieu. Thus we can use plasmid vectors designed to produce a hybrid protein containing an efficient signal peptide coupled to the amino terminus of the cloned heterologous protein (secretion cloning vectors) for the production of proteins which are insoluble, proteolytically sensitive, or bacteriocidal when produced in the cytoplasm of Escherichia coli. We demonstrate that human granulocyte-macrophage colony stimulating factor can be isolated as an active species only after transport into the bacterial periplasm. Production of the protein in the bacterial cytoplasm is bacteriocidal. We also demonstrate that biologically active human interleukin 4 appears only after transport of the protein into the bacterial growth medium. The protein forms membrane-associated aggregates in the cytoplasm, and demonstrates an active but nonnative conformation when expressed in the periplasm. This may correlate with the affinity of the interleukin 4 molecule for negatively charged macromolecules, including bacterial membrane components and bacterial lipopolysaccharides, which may alter the folding pathway inside the cell.     

Effect of some carbamate pesticides on nodulation,plant yield and nitrogen fixation byPisum sativum andVigna sinensis in the presence of their respective rhizobia

Summary Six carbamate pesticides namely 1-naphthol, sevin, dimetilan, trematan, NaDDC and dymid were studied to see their effect on nodulation and nitrogen fixation inPisum sativum andVigna sinensis. Low concentrations of the pesticides have little effect on nodulation and nitrogen fixation, whereas higher concentrations adversely effect these processes. The results also indicate that then sensitivity depends upon the species of the Rhizobium and also the type of the pesticide. Pesticides belonging to the carbamate group differ in their capacity to affect nodulation and nitroge fixation.     

Inhibition of S-adenosyl-L-methionine sterol-C-24-methyltransferase by analogues of a carbocationic ion high-energy intermediate. Structure activity relationships for C-25 heteroatoms (N, As, S) substituted triterpenoid derivatives

Microsomes from maize seedlings are capable of catalyzing the C-24 alkylation of 4,4,14 alpha-trimethyl-9 beta,19-cyclo-5 alpha-cholest-24-en-3 beta-ol (cycloartenol) by (S)-adenosyl-L-methionine (AdoMet) leading to 24-methylene cycloartanol. Derivatives of cycloartenol bearing a nitrogen atom at C-25 have been previously shown to be potent inhibitors of the AdoMet-cycloartenol-C-24-methyltransferase (Narula, A. S., Rahier, A., Benveniste, P., and Schuber, F. (1981) J. Am. Chem. Soc. 103, 2408-2409). In order to determine the molecular parameters of the inhibition and to gain information about its mechanism, various azasteroids and analogues have been synthesized and assayed. The following results have been obtained. i) The presence of a positive charge at position 25 was found to be the major cause of the inhibition since electrostatically neutral isosteric compounds possessing a carbon in place of the nitrogen atom were not inhibitory. The positive charge leading to inhibition may be conferred by a protonated amine, a quaternary ammonium group, as well as by a sulfonium or an arsonium group. ii) A steroid-like structure of the inhibitor was also important. And iii) the presence of a free 3 beta-hydroxy group and the bent conformation of cycloartenol, which are essential molecular features of the substrate for the methylation reaction, were no longer required to observe inhibition. The data obtained strongly support the idea that C-25 heteroatoms (N, As, and S), substituted triterpenoid derivatives possessing a positive charge at position 25, are analogues of a carbocationic high-energy intermediate involved during the reaction catalyzed by the AdoMet-cycloartenol-C-24-methyltransferase.     

Identification and characterization of bacterial endophytes of rice

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.