The binding of the retro-analogue of glutathione disulfide to glutathione reductase

The retro-analogue of glutathione disulfide was bound to the GSSG binding site of crystalline glutathione reductase. The binding mode revealed why the analogue is a very poor substrate in enzyme catalysis. The observed binding mode difference between natural substrate and retro-analogue is explained.     

Biological and molecular variability of human immunodeficiency virus type 2 isolates from The Gambia.

Seven new human immunodeficiency virus type 2 (HIV-2) isolates (CBL-20 to CBL-26) from The Gambia were characterized. Their cytopathogenicity and growth in vitro correlated with the severity of clinical disease. CBL-22 was highly sensitive to neutralization by HIV-2 sera and was cross-neutralized by some HIV-1 sera. These findings, the differing sizes of envelope glycoproteins of individual isolates, and the sequence analysis of amplified regions of the viral DNAs show that these HIV-2 isolates from one geographical region in West Africa exhibit biological and genome variability comparable to that observed for HIV-1.     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Characterization and purification of membrane-associated phosphatidylinositol-4-phosphate kinase from human red blood cells

The membrane-bound form of phosphatidylinositol-4-phosphate (PtdInsP) kinase was purified 4,300-fold from human red blood cells to a specific activity of 117 nmol min-1 mg-1. Although this enzyme copurified with red blood cell membranes, it was solubilized by high salt extraction in the absence of detergent indicating that it is a peripheral membrane protein. The major protein seen in the most purified preparation migrated at 53,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major PtdInsP kinase activity in this preparation was also coincident with this 53,000-dalton band upon renaturation of activity from SDS-PAGE. To test further whether the 53,000-dalton protein contained PtdInsP kinase activity, antibodies were prepared against the gel-purified 53,000-dalton protein. This antiserum was able to precipitate both the 53,000-dalton peptide and PtdInsP kinase activity from red blood cell membranes. The apparent size of the native enzyme in the most purified preparation was determined to be 150,000 +/- 25,000 daltons by gel filtration. This PtdInsP kinase activity was at least 100-fold more active in phosphorylating PtdInsP than phosphatidylinositol and was easily separated from the red cell membrane phosphatidylinositol kinase by salt extraction. Analysis of the reaction product, phosphatidylinositol 4,5-bisphosphate, indicates that the enzyme phosphorylates phosphatidylinositol 4-phosphate specifically at the 5'-hydroxyl of the inositol ring. The apparent Km for ATP was 2 microM, and the concentrations of Mg2+ and Mn2+ giving half-maximal activity were 2 and 0.2 mM, respectively. Mg2+ supported 3-fold higher activity than Mn2+ at optimal concentrations. The enzymatic activity was inhibited by its product, phosphatidylinositol 4,5-bisphosphate and enhanced by phosphatidylserine.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli

We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin [L-homoalanine-4-yl-(methyl)phosphinic acid], the active ingredient of the herbicide Basta (Hoechst AG). The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment. The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source. The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E. coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E. coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19). A number of expression plasmids carrying the isolated transaminase gene were constructed. With these constructs, the transaminase expression in transformants of E. coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria. Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor.     

Isoelectric focusing analysis of detergent extracted renal 11 beta-hydroxysteroid dehydrogenase

11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.     

Molecular cloning,nucleotide sequence and expression in Escherichia coli of the β-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8

Summary The -cyclodextrin glycosyltransferase (-CGTase) gene was isolated from a -library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant -CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme. Offprint requests to: H. Bender     

Molecular size characterization of bacterial capsular polysaccharide vaccines by high performance liquid chromatography

Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.     

Alveolar slope and dead space of He and SF6 in dogs: comparison of airway and venous loading

Series (Fowler) dead space (VD) and slope of the alveolar plateau of two inert gases (He and SF6) with similar blood-gas partition coefficients (approximately 0.01) but different diffusivities were analyzed in 10 anesthetized paralyzed mechanically ventilated dogs (mean body wt 20 kg). Single-breath constant-flow expirograms were simultaneously recorded in two conditions: 1) after equilibration of lung gas with the inert gases at tracer concentrations [airway loading (AL)] and 2) during steady-state elimination of the inert gases continuously introduced into venous blood by a membrane oxygenator and partial arteriovenous bypass [venous loading (VL)]. VD was consistently larger for SF6 than for He, but there was no difference between AL and VL. The relative alveolar slope, defined as increment of partial pressure per increment of expired volume and normalized to mixed expired-inspired partial pressure difference, was larger by a factor of two in VL than in AL for both He and SF6. The He-to-SF6 ratio of relative alveolar slope was generally smaller than unity in both VL and AL. Whereas unequal ventilation-volume distribution combined with sequential emptying of parallel lung regions appears to be responsible for the sloping alveolar plateau during AL, the steeper slope during VL is attributed to the combined effects of continuing gas exchange and ventilation-perfusion inequality coupled with sequential emptying. The differences between He and SF6 point at the contributing role of diffusion-dependent mechanisms in intrapulmonary gas mixing.