Regulation of the phosphate regulon of Escherichia colt Properties of phoR deletion mutants and subcellular localization of PhoR protein

Summary The phoR gene is a bifunctional regulatory gene for the phosphate regulon of Escherichia coli. It acts as a negative regulator in the presence of excess phosphate and as a positive regulator with limited phosphate, through modification of PhoB protein. We constructed several phoR genes, with various deletions in the 5 regions, which were regulated by the trp-lac hybrid promoter. The PhoR1084 and PhoR1159 proteins that lack the 83 and 158 N-terminal amino acids, respectively, retained the positive function for the expression of phoA that codes for alkaline phosphatase, but lacked the negative function. The PhoR1263 protein that lacks the 262 N-terminal amino acids was deficient in both functions. An antiserum against PhoR1084 protein was prepared. Western blot analysis of the subcellular fractions obtained by differential centrifugation indicated that the intact PhoR and PhoR1084 proteins are located in the inner membrane and cytoplasmic fractions, respectively. The results suggest that PhoR protein is anchored to the cytoplasmic membrane by the amino-terminal region.     

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. Brian Reavy and Svetlana Bagirova contributed equally to this work.     

Probing FhuA''-''PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12

Summary The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%–90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA-PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.     

Alkaline phosphatase which lacks its own signal sequence becomes enzymatically active when fused to N-terminal sequences of Escherichia coli haemolysin (HlyA)

Summary Fusion of the alkaline phosphatase gene (phoA) which lacks its own signal peptide sequence to the N-terminal region of hlyA, the structural gene for Escherichia coli haemolysin, leads to active alkaline phosphatase (AP). AP activity depends on the length of the N-terminal region of hlyA. An optimum is reached when 100–200 amino acids of HlyA are fused to PhoA but fusion of as little as 13 amino acids of HlyA to PhoA is sufficient to yield appreciable AP activity. When cells are treated with lysozyme most of the AP activity is found associated with the membrane fraction but a substantial amount is also found in the soluble fraction, most of which may represent, a periplasmic pool of AP. The soluble portion of AP activity is significantly increased when the cells are disrupted by ultrasonication, which indicates that the fusion proteins are only loosely associated with the membrane and that large parts are already located on the outside of the cytoplasmic membrane. The expected fusion proteins were identified in the soluble and the membrane fractions and their amounts in these fractions correlated well with AP activity.     

Effect of phosphorus supply on phosphate uptake and alkaline phosphatase activity in Rhizobia

The effect of P nutrition on phosphate uptake and alkaline phosphatase activity was studied in chemostat culture for four rhizobial and three bradyrhizobial species. Phosphate-limited cells took up phosphate 10- to 180-fold faster than phosphate-rich cells. The four fast-growing rhizobial strains contained high levels of alkaline phosphatase activity under P-limited conditions compared to the repressed levels found in P-rich cells; alkaline phosphatase activity could not be detected in three slow-growing rhizobial strains, regardless of their P-status.Glycerol 1-phosphate-uptake in the cowpea Rhizobium NGR234 was derepressed over 50-fold under P-limited conditions, and appeared to be co-regulated with phosphate uptake.The phosphate-uptake system appeared similar in all strains with apparent K _m values ranging from 1.6 M to 6.0 M phosphate and maximum activities from 17.2 to 126 nmol · min^-1 · (mg dry weight of cells)^-1. Carbonyl cyanide m-chlorophenyl hydrazone strongly inhibited phosphate uptake in all strains and a number of other metabolic inhibitors also decreased phosphate uptake in the cowpea Rhizobium NGR234. The phosphate uptake system in all strains failed to catalyse exchange of ³²P label in preloaded cells or efflux of phosphate. The results suggest a single, repressible, unidirectional and energy-dependent system for the transport of phosphate into rhizobia.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanesulphonic acid     

Adaptation of intestinal cell membrane enzymes to low temperatures in the Antarctic teleost Pagothenia bernacchii

The enzymatic activity (expressed as milliunits per milligram total proteins) of three intestinal brush-border membrane enzymes, leucine aminopeptidase, alkaline phosphatase and maltase, measured over a range of temperatures between 1.5 and 37 °C, has been found to be much higher in the Antarctic fish Pagothenia bernacchii than in the temperate fish Anguilla anguilla. To explain this experimental observation the apparent Michaelis-Menten constant, the maximal velocity, the activation energy values and the thermal stability of these three enzymes were measured. The apparent Michaelis-Menten constant values of leucine amino peptidase and alkaline phosphatase were different in the intestine mucosal homogenate of the two fish at each measured temperature (from a minimum of 2.5 to a maximum of 37 °C). However, the values found at 2.5 °C for the Antarctic species and 15 °C for the eel where comparable. Furthermore, its value was unchanged in eel intestine apical membranes, both in the presence and without enzyme lipid microenvironment. While the maximal enzymatic activities of the leucine aminopeptidase and maltase did not decrease without their enzyme lipid microenvironment, produced by treatment with Triton X-100, the impairment of alkaline phosphatase maximal activity cannot be significantly differentiated from a non-specific inhibitory effect of the detergent. The activation energy values of leucine amino peptidase, alkaline phosphatase and maltase were lower in the Antarctic fish (11.7, 5.6 and 11.8 kcal·mol^-1, respectively) than in the eel (13.6, 7.6 and 13.1 kcal·mol^-1, respectively). The thermal stability of alkaline phosphatase and maltase is different in Pagothenia bernacchii and Anguilla anguilla intestinal homogenate.Abbreviations BBM brush border membrane - E _a activation energy - EGTA ethyleneglycol-bis-(-amino ethylether)N, N-tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethane sulphonic acid - Km_app apparent Michaelis-Menten constant - PMSF phenylmethyl-sulphonyl fluoride - TRIS TRIS (hydroxymethyl)-aminomethane     

A continuous culture study of the phosphorus nutrition of Rhizobium trifollii WU95, Rhizobium NGR234 and Bradyrhizobium CB756

With continuous cultures in a fully defined minimal salts medium steady states were achieved at both limiting and non-limiting concentrations of phosphate in the inflowing medium for Rhizobium trifolii WU95, cowpea Rhizobium NGR234, and Bradyrhizobium CB756.Millimolar growth yields obtained from P-limited cultures varied over 2-fold from 3.2 g dry weight·(mmol P)^-1 for WU95 to 5.3 g dry weight·(mmol P)^-1 for CB756 and 7.2 g dry weight·(mmol P)^-1 for NGR234.For both WU95 and NGR234 growth under P-excess conditions resulted in elevated levels of total biomass P and the storage compound polyphosphate, compared with P-limited cultures. However, P-limited cultures of these two strains still contained significant quantities of polyphosphate. The P-status for CB756 cultures did not affect either total biomass P or polyphosphate levels. Alkaline phosphatase was maximally derepressed in P-limited cultures of WU95 and NGR234. However, in CB756 alkaline phosphatase was not detected at significant levels regardless of its P supply.These data suggest that growth of rhizobia is controlled predominantly by the attainment of a critical internal P level.Abbreviation HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of plants by the pTF-FC2 plasmid is efficient and strictly dependent on the MobA protein

In the transformation of plants by Agrobacterium tumefaciens the VirD2 protein has been shown to pilot T-DNA during its transfer to the plant cell nucleus. Other studies have shown that the MobA protein of plasmid RSF1010 is capable of mediating its transfer from Agrobacterium cells to plant cells by a similar process. We have demonstrated previously that plasmid pTF-FC2, which has some similarity to RSF1010, is also able to transfer DNA efficiently. In this study, we performed a mutational analysis of the roles played by A. tumefaciens VirD2 and pTF-FC2 MobA in DNA transfer-mediated by A. tumefaciens carrying pTF-FC2. We show that MobA+/VirD2+ and MobA+/VirD2– strains were equally proficient in their ability to transfer a pTF-FC2-derived plasmid DNA to plants and to transform them. However, the MobA–/VirD2+ strain showed a DNA transfer efficiency of 0.03% compared with that of the other two strains. This sharply contrasts with our results that VirD2 can rather efficiently cleave the oriT sequence of pFT-FC2 in vitro. We therefore conclude that MobA plays a major VirD2-independent role in plant transformation by pTF-FC2.     

Characterization of three membrane fractions isolated from cells of Rhodopseudomonas capsulata adapting from chemotrophic to phototrophic conditions

By means of sucrose density centrifugation three membrane fractions, named light, medium and heavy have been isolated from cells of Rhodopseudomonas capsulata strain 37b4, adapting from chemotrophic to phototrophic growth conditions. Succinate dehydrogenase activity of aerobically grown cells was mainly confined to the heavy (chromatophore) fraction. Upon changing to phototrophic conditions the activity of the succinate dehydrogenase increased in the medium and light fraction. All fractions contain bacteriochlorophyll. NADH dehydrogenase of chemotrophically grown cells was enriched in the light and medium fraction but is increased in the heavy fraction under phototrophic growth conditions. The capacity of photophosphorylation is high in the light and heavy fraction. The results indicate a differentially incorporation of functional subunits into specific parts of the membrane system during membrane differentiation.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Isolation of plasma membrane complexes fromXenopus oocytes

Summary A method for the isolation of plasma membrane fractions fromXenopus oocytes has been developed, and the membranes have been characterized biochemically and morphologically. Plasma membrane complexes prepared by this procedure consisted of large sheets of the membrane, with associated vitelline envelope (a nonmembranous meshwork of fibers) and cortical (secretory) granules still attached. The morphology of cell surface microvilli and coated pits was well preserved. Cortical granules were removed by gentle homogenization in a low ionic strength medium, and integral and peripheral membrane proteins were then separated from vitelline envelopes by detergent extraction and phase separation in Triton-X-114. Biochemical characterization of the plasma membrane fractions indicated substantial levels of 5-nucleotidase and alkaline phosphodiesterase activity associated with the oocyte cell surface, with 44–66% recovery of these markers in the final membrane preparations. Lectin blotting and lectin affinity chromatography with Concanavalin A and wheat germ agglutinin were used to characterize the major glycoprotein species associated with the plasma membrane complexes. Plasma membrane fractions prepared by this procedure should be very useful in both biochemical and morphological studies of membrane protein sorting in theXenopus oocyte system.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Composition of axolemma-enriched fractions isolated from bovine CNS myelinated axons

Axolemma-enriched fractions were isolated from the white matter of bovine corpus callosum via a purified preparation of myelinated axons which were osmotically shocked and fractionated on a discontinuous density gradient. Two membrane fractions of differing density were obtained; both were somewhat enriched over white matter whole homogenate in specific activity of acetylcholinesterase and 5-nucleotidase and maximal binding capacity for saxitoxin. Both membrane fractions contained appreciable amounts of 2, 3-cyclic nucleotide 3-phospho-hydrolase; the specific activity of antimycin-sensitive NAPH-cytochromec reductase and cytochromec oxidase indicated low levels of contamination by microsomal and mitochondrial membrane. The myelin which is concomitantly isolated with the axolemma-enriched fractions has a lipid and protein composition comparable to that of myelin isolated by other procedures. Both axolemma-enriched fractions contain about one half of their dry weight as lipid comprised of approximately 25% cholesterol, 25% galactolipid (cerebrosides and sulfatides in a molar ratio of about 4:1) and 50% phospholipid, mostly choline phosphatides and ethanolamine phospholes in an equimolar ratio. The axolemma fractions are also enriched in ganglioside content relative to the myelin fraction. The polypeptides of the axolemma-enriched fractions range from 20,000 to over 200,000 in molecular weight; the predominant proteins are in the range from 50,000 to 69,000. The most dense axolemma-enriched fraction is over fourfold enriched in glyco-protein content compared with myelin, with at least 10 different molecular-weight classes of glycoproteins as identified by Schiff stain of polyacrylamide gel protein profiles. The differences and similarities in the molecular composition of axolemma-enriched preparations which have been characterized to date are discussed.     

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis

Summary The ptsG gene of Bacillus subtilis encodes Enzyme II^G1c of the phosphoenolpyruvate: glucose phosphotransferase system. The 3 end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes III^Glc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5 end. These results, combined with the sequence of the 3 end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes II^Glc and II^Nag of E. coli but which are present in the II^G1c-like protein encoded by the E. coli malX gene.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

Molecular cloning of the cDNA encoding a stress-inducible protein phosphatase 1 (PP1) catalytic subunit from French bean (Phaseolus vulgaris L.)

A cDNA showing high sequence similarity (>70%) to plant protein phosphatase 1 catalytic subunit variants from other species has been isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. The clone appears to be a near full-length 1431 bp with a 172 bp 5-untranslated region and a 317 bp 3-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with predicted M _r of 35552. Alternatively an ATG situated to the 5 end of the putative start site would increase the protein size by 6 amino acids.The mRNA for Pvpp1 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean. The cloned cDNA represents one of the few examples of a gene product that is probably involved in dephosphorylation events arising after the initial responses to biotic stress.Abbreviations PAL phenylalanine ammonia-lyase - PP1 protein phosphatase 1 - Pvpp1 Phaseolus vulgaris protein phosphatase 1     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens

Abstract The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter, Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli . Because Osa inhibits oncogenicity of A. tumefaciens , and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.     

Specific binding of the calcium channel blocker [3H]verapamil to membrane fractions of Chlamydomonas reinhardtii

Specific binding of the calcium antagonist [³H]verapamil to a microsomal fraction, a presumptive plasma membrane fraction and an intracellular membrane fraction of the phototactic unicellular green alga Chlamydomonas reinhardtii has been demonstrated. The specific activity of the plasma membrane marker enzyme K⁺-stimulated, Mg²⁺-dependent ATPase was severalfold higher in the upper (polyethylene glycol-rich) than in the lower (dextran-rich) phase, and the reverse was established for the marker enzymes of intracellular membranes such as cytochrome c oxidase for mitochondria and antimycin Aresistant NADPH-cytochrome c reductase for endoplasmic reticulum. Chlorophyll as a marker for thylakoid fragments was exclusively found in the lower phase. In the microsomal fraction two specific binding sites of [³H]verapamil were found at 22°C, one with higher and a second with lower affinity to [³H]verapamil. Separation of plasma membranes from intracellular membranes revealed that the highaffinity binding site is attributed to the plasma membrane fraction whereas the low-affinity binding site can be attributed to the intracellular membrane fraction. Specific binding to both separated membrane fractions is saturable and reversible. [³H]Verapamil binding to plasma membranes was not inhibited by the calcium channel blockers diltiazem and nifedipine. However, in the intracellular membrane fraction [³H]verapamil could be displaced by diltiazem but not by nifedipine. Increasing concentrations of calcium chloride inhibited [³H]verapamil binding in both fractions.Abbreviations B_max maximum density of binding sites - BSA bovine serum albumin - Cyt.c cytochrome c - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-amino-ethylether)N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IC₅₀ concentration causing 50% inhibition - Mes [N-morpholino]ethanesulfonic acid - PEG polyethylene glycol - PMSF phenylmethylsulfonylfluoride - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid