Interaction of extracellular Pseudomonas lipase with alginate and its potential use in biotechnology

Summary Extracellular Pseudomonas lipase is able to interact directly or indirectly with alginate as deduced from the following results: (i) During adsorption chromatography of exolipase the enzyme adsorbed quantitatively to glass beads in the absence of alginate, but not after its preincubation in the presence of the polysaccharide; pretreatment of glass beads with alginate did not prevent enzyme adsorption. (ii) In the presence of alginate exolipase was much more resistant to heat inactivation than in its absence. (iii) In the presence of alginate the increase in exolipase activity caused by the non-ionic detergent Triton X-100 was drastically reduced. (iv) Exolipase could be rapidly and almost completely harvested from cell-free culture fluid of P. aeruginosa 5940 by ethanolic coprecipitation with alginate. After dissolving the coprecipitate in detergent-containing buffer exolipase and polysaccharide could be easily separated by ion-exchange chromatography on DEAE-Sephadex A-25. The coprecipitation method was also successfully applied to exolipases produced by Pseudomonas sp., Chromobacierium viscosum and Rhizopus delamar, thus suggesting potential use of this method in biotechnology.     

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties.

Alginate-producing (mucoid) strains of Pseudomonas aeruginosa possess a 54-kDa outer membrane (OM) protein (AlgE) which is missing in nonmucoid bacteria. The coding region of the algE gene from mucoid P. aeruginosa CF3/M1 was subcloned in the expression vector pT7-7 and expressed in Escherichia coli. The level of expression of recombinant AlgE was seven times higher than that of the native protein in P. aeruginosa. Recombinant AlgE was found mainly in the OM. A putative precursor protein (56 kDa) of AlgE could be immunologically detected in the cytoplasmic membrane (CM). Surface exposition of AlgE in the OM of E. coli was indicated by labeling lysine residues with N-hydroxysuccinimide-biotin. Secondary-structure analysis suggested that AlgE is anchored in the OM by 18 membrane-spanning beta-strands, probably forming a beta-barrel. Recombinant AlgE was purified, and isoelectric focusing revealed a pI of 4.4. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels with a single-channel conductance of 0.76 nS in 1 M KCl and a mean lifetime of 0.7 ms. Single-channel current measurements in the presence of other salts as well as reversal potential measurements in salt gradients revealed that the AlgE channel was strongly anion selective. For chloride ions, a weak binding constant (Km = 0.75 M) was calculated, suggesting that AlgE might constitute an ion channel specific for another particular anion, e.g., polymannuronic acid, which is a precursor of alginate. Consistent with this idea, the open-state probability of the channel decreased when GDP-mannuronic acid was added. The AlgE channel was inactivated when membrane voltages higher than +85 mV were applied. The electrophysiological characteristics of AlgE, including its rectifying properties, are quite different from those of typical porins.     

Broadly reactive murine cytotoxic cells induced in vitro under syngeneic conditions

Mouse spleen cells became cytotoxic in short-term 51Cr-release assays for a wide variety of target cells after 5 days of culture in vitro with polyinosinic acid in a system that was otherwise entirely syngeneic. This study characterizes these effector cells with respect to target specificity, effect of donor age, time course of their appearance, mouse strain differences, and expression of differentiation antigens Thy-1, Lyt-1, Lyt-2, NK-1, and asialo GM1. The combination of properties of this cytotoxic cell response that make it unique are that a) the broadly reactive cytotoxic activity developed from unprimed spleen cells in the absence of either foreign cells or foreign serum; b) the response did not peak until 4 to 5 days of culture in vitro; c) the broad reactivity pattern included freshly dispersed primary syngeneic sarcoma cells and cultured syngeneic fibroblasts but did not include syngeneic lymphoblast target cells; d) the response was largely monoclonal as defined by target cell binding; and e) cytotoxic cell activity was sensitive in complement-mediated treatments to both anti-NK and anti-theta but not to anti-Lyt-2, anti-Lyt-1, or anti-asialo GM1. Both high- and low-responding mouse strains have been identified.     

Effect of fat free diet during the last week of pregnancy upon the linoleic and arachidonic acid content of fetal organ lipids and placenta lipids of the rat

Pregnant Wistar rats were fed a fatfree diet from day 16--22 of pregnancy. On day 22, the fatty acid components of cholesterol esters, triglycerides, free fatty acids and phospholipids of maternal (brain, muscle, serum, white adipose tissue, liver) and fetal (brain, carcass, serum, liver) tissues, including the placenta, were examined gaschromatographically for the participation of linoleic and arachidonic acid. In all fetal and maternal organs the linoleic acid levels in the fatty acid patterns were strongly reduced. The alterations nearly always involved all the lipid fractions of a tissue and were mostly equal within a tissue. The strongest decreases of linoleic acid occurred in the placenta, and the weakest, in the lipids of maternal muscle and maternal adipose tissue. The linoleic acid alterations were principally similar in fetal and the corresponding maternal tissues, while being less pronounced in case of maternal muscle. The participation of arachidonic acid in the fatty acid pattern is completely retained in the lipids of fetal organs, and is even enhanced in those of the placenta.     

Pyruvate kinase “Göttingen1,2”: Congenital hemolytic anemia,evidence of double heterozygosity,and lack of enzyme cooperativity

Summary Double heterozygosity of pyruvate kinase (PK) deficiency associated with hereditary hemolytic anemia is emphasized by studies of a kindred harboring two distinct mutant forms of this enzyme. The hematologically unaffected parents exhibit slightly reduced PK activity, a normal Hill coefficient, and a normal thermodynamic dissociation constant for the overall reaction. The paternal enzyme is characterized by normal substrate affinities and decreased activities with the substrate analogues CDP and GDP, whereas the maternal enzyme shows normal affinity for PEP, but an increased affinity for ADP and low thermostability. It is assumed that the erythrocytes of the parents contain a mixture of normal PK and a functionally abnormal isoenzyme, the latter differing between the parents. The two children suffer from hereditary hemolytic anemia. Their PK must be a combination of the mutant paternal and maternal isoenzymes, and their activities are reduced to about 30%. These enzymes are characterized by an increased affinity for PEP and a decreased affinity for ADP, a Hill coefficient of about 1 (indicating lack of cooperativity due to a loss of its allosteric properties), a decreased overall catalytic activity, and a higher resistance to heat denaturation. Further differences are observed in the SDS-gel electrophoresis between the two patients' enzymes. From the enzymological point of view it is impossible to characterize true PK variants in such double heterozygous cases which contain a combination of two different isoenzymes. The cause of chronic hemolysis appears to depend mainly on the loss of the allosteric properties, i.e., the lack of enzyme cooperativity.