Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Effects of UV-B radiation and nitrogen starvation on enzyme activities in isolated plasma membranes of Euglena gracilis

Circadian rhythms are characteristic of many physiological and biochemical processes in the freshwater flagellate Euglena gracilis. Earlier, we found that the rhythms of photosynthesis, phototaxis and cell shape followed the same pattern in control organisms, but were differently affected by stress such as UV-B irradiation and nitrogen deficiency. Here we extend our studies to use isolated plasma membranes to characterize the rhythms of some plasma membrane-bound enzymes. Also, we wanted to see whether stress-induced changes of these rhythms could be detected at the subcellular level and possibly be coupled to the changes seen in photosynthesis, phototaxis and cell shape. The isolation of plasma membranes using aqueous polymer two-phase partitioning was successful, as judged by the large enrichment of the plasma membrane-marker 5′-nucleotidase, and the difference in the polypeptide pattern compared with the microsomal fraction from which it was prepared. Two other enzymes were analyzed, K⁺, Mg²⁺-ATPase, and adenylyl cyclase. The specific activities of all three enzymes were decreased by UV-B radiation by ca 30–50%, compared with the control cultures. On the other hand, nitrogen deficiency not only reduced the activity of the K⁺.Mg²⁺-ATPase but also increased the activities of the 5′-nucleotidase and adenylyl cyclase. The different treatments also resulted in differences in polypeptide pattern, e.g., a polypeptide around 30 kDa seemed to be specific to plasma membranes of nitrogen-deficient cultures and one at 39 kDa for the UV-B radiated ones. All three enzymes showed diurnal rhythms that were affected by UV-B radiation. The peak in the rhythm of the ATPase was shifted by UV-B radiation, the rhythm of the 5′-nucleotidase nearly eliminated. The first peak of adenylyl cyclase activity was delayed, so that it looked more like a broad peak between 2 and 11 h after the onset of light. The rhythm of ATPase activity could be correlated with that of photosynthesis in both control and UV-B irradiated cultures. Also, the rhythms of adenylyl cyclase activity and cell shape changes showed some similarities.     

Chromatographic resolution of some cyclic sulfoximides derived from prochiral and chiral sulfoxides

Three endocyclic sulfoximides of the 1-aryl- and 1-alkyl-3-oxo-benzo[d]-isothia (IV)-azole 1-oxide type (1-substituent = 2′-carboxyphenyl, 2′-carbethoxyphenyl, and octyl, respectively) were found to be well resolved on a chiral phase derived from bovine serum albumin (BSA). Selectivities (α) of 1.74, 1.12, and 1.44, respectively, were obtained. The retention behaviour of 1-octyl-3-oxo-benzo[d]isothia(IV)-azole 1-oxide was further investigated in some detail as a function of the mobile phase composition and the elution order was established from optically active material obtained from the enantiopure sulfoxide precursor. An enantiomeric excess of 85.4% was obtained in the cyclocondensation reaction of the octyl-substituted sulfoxide precursor with hydrazoic acid to the corresponding endocyclic sulfoximide. © 1995 Wiley-Liss, Inc.     

Topology of the membrane-associated hepatitis C virus protein NS4B

Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in FLAVIVIRIDAE:     

Actin-like protein associated with plasma membranes fromEuglena gracilis

Summary Microtubules are characteristic components of the membrane skeleton ofEuglena gracilis, but whether microfilaments are present has been controversial. We here present evidence that an actin-like protein may indeed be associated with the plasma membrane (PM) ofE. gracilis. Firstly, a 47 kDa, PM-associated, polypeptide was recognized by an anti-amoeba actin antibody. Secondly, this 47 kDa protein seemed to be peripherally attached to PM in much the same way as -tubulin, since both could be released from PM by treatment with 150 mM NaOH but not with ethylene glycol, NaCl, or formamide. Thirdly, the 47 kDa polypeptide and -tubulin were found mainly in the Triton X-1 14-insoluble fraction, indicating that they were part of a protein complex resistant to detergents, such as the cytoskeleton. Finally, DNase I activity was inhibited by a fraction enriched in the 47 kDa polypeptide, a property typical of actin.Abbreviations CP-medium cytoskeleton preparation medium - BNSP-skatole 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine - ECL enhanced chemiluminescence - HEPES N-[2-hydroxyethyl]-piperazine-N[2-ethane sulfonic acid] - ICM intracellular membranes - MF mitochondrial and microsomal fraction - PM plasma membrane - PPB potassium phosphate buffer - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - TBS Tris-buffered saline - TBST Tris-buffered saline with Tween 20     

<Emphasis Type="Italic">Trichoderma viride</Emphasis> cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells

Background  

Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation.     

Characterization of prolamellar bodies, from dark-grown seedlings of Scots pine, containing light- and NADPH-dependent protochlorophyllide oxidoreductase

Cotyledons of conifers have a light-independent pathway for chlorophyll biosynthesis. To investigate whether the prolamellar body of Scots pine ( Pinus sylveslris L.) is similar to the better known prolamellar body of wheat, etioplast membrane fractions were isolated from cotyledons of dark-grown Scots pine. Dark-grown cotyledons contained both chlorophyll and protochlorophyllide, 158 and 10 nmol (g fresh weight)'respectively, and had a chlorophyll a to b ratio of 4.2. The content of glyco- and phospholipids was 7.1 μmol (g fresh weight)¹. About 40 mol % of these lipids were the specific plastid lipids – monogalactosyl diacylglycerol. digalactosyl diacylglycerol and sulfoquinovosyl diacylglycerol in the relative amounts 50, 35 and 7 mol %. The mol ratio of monogalactosyl diacylglycerol to digalactosyl diacylglycerol was 1.7. Low temperature fluorescence emission spectra of intact cotyledons and homogenate showed maxima at 633, 657, 686, 696 nm and a broad peak at 725–735 nm. The maxima at 633 and 657 nm represented different forms of protochlorophyllide and the other emission maxima represented chlorophyll protein complexes. The 657 nm form of protochlorophyllide was phototransformable both in vivo and in the isolated membranes. The phototransformable protochlorophyllide was substantially enriched in the prolamellar body fraction.
The specific activity of light dependent protochlorophyllide oxidoreductase in the prolamellar body fraction was found to be 2 nmol chlorophyllide formed [(mg protein)⁻¹ min⁻¹]. The molecular weight of the enzyme polypeptide was determined as 38 000 dalton with sodium dodecylsulphate-polyacrylamide gel electrophoresis.