A protein with lectin activity in penetration glands of Diplostomum pseudospathaceum cercariae

Homogenates of Diplostomum pseudospathaceum cercariae agglutinated mouse erythrocytes. The haemagglutination could be inhibited by certain glycoconjugates containing beta-1,3- and beta-1,4-glycan chains and also by some simple saccharides. The most potent inhibitors were heparin and some other glycosaminoglycans, bacterial lipopolysaccharides, laminarin (a beta-1,3-glucan) and lactulose. After electrophoresis of cercarial proteins, a dominant double band appeared in the 22-24 kDa region of gels. On blots, this protein bound labelled laminarin and it was also one of the few proteins recognised by mouse antibodies raised against cercarial haemagglutinins. In addition, mouse polyclonal antibodies against the beta-1,3-glucan-binding protein bound exclusively to the 22-24 kDa region on Western blots. Histochemistry revealed strong binding of labelled laminarin to cercarial penetration glands; this reaction was fully blocked by unlabelled laminarin. Other labelled glycoconjugates such as heparin, hyaluronic acid and a bacterial lipopolysaccharide also bound to the glands. Immunohistochemistry confirmed the localisation of the beta-1,3-glucan-binding protein in penetration glands. Reaction of the cercarial protein with immunoglobulins from non-immunised mice was observed on both nitrocellulose membranes and histological sections; this could be blocked by laminarin in incubation buffers. We consider the cercarial haemagglutinin to be a lectin which is identical with the 22-24 kDa beta-1,3-glucan-binding protein. According to the binding specificity and localisation we speculate on a role of this lectin in cercarial penetration into the host, probably as a tissue recognition or antibody rendering factor.     

A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.     

The structure of the hepatic insulin receptor and insulin binding.

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.     

Identification of Na+,Pi-binding protein in kidney and intestinal brush-border membranes.

An Na+, Pi-binding protein has been extracted from kidney and intestinal brush-border membranes with an organic solvent and has been purified by Kieselghur and Sephadex LH-60 chromatography. The molecular mass of this protein has been estimated to be about 155 kDa as determined by gel-filtration chromatography on Sepharose 2B. Under denaturing conditions, polyacrylamide-gel electrophoresis revealed a monomer of molecular mass about 70 kDa. The protein has high specificity and high affinity for Pi [K0.5 (concentration at which half-maximal binding is observed) near 10 microM]. Na2+ binding also exhibits saturation behaviour, with a K0.5 near 7.5 mM. Pi binding is inhibited by known inhibitors of Pi transport in brush-border membrane vesicles. It appears that this protein could be involved in Na+/Pi co-transport across the renal and intestinal brush-border membranes.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation.

A heparin-binding protein was isolated from bovine uteri and purified to homogeneity. This protein appears as a double band of approx. 78 kDa in SDS/polyacrylamide-gel electrophoresis and has an isoelectric point of 5.2. The binding of heparin to this protein is saturable. No other glycosaminoglycan from mammalian tissue, such as hyaluronic acid, chondroitin sulphate, dermatan sulphate or keratan sulphate, binds to the 78 kDa protein. Dextran sulphate binds in a non-saturable fashion. Certain heparan sulphate polysaccharide structures are required for binding to the 78 kDa protein. Some proteoheparan sulphates, such as endothelial cell-surface proteoheparan sulphate, show only weak interaction with the 78 kDa protein in contrast with a basement-membrane proteoheparan sulphate from HR-9 cells. Antibodies against the 78 kDa protein inhibit binding of proteoheparan [35S]sulphate from basement membranes to smooth-muscle cells. Conventional antibodies, Fab fragments and some monoclonal antibodies, inhibit smooth-muscle cell proliferation in a similar range as that observed for heparin. The protein was detected in a variety of tissues and cells but not in blood cells. A possible role of this protein as a receptor for heparin or heparan sulphate and its function in the control of the arterial wall structure are discussed.     

Purification and characterization of the adenosine A2-like binding site from human placental membrane

We have purified and characterized the adenosine A2-like binding site from human placental membranes. 5'-N-Ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binds to this site, with a Kd of 240 nM and a Bmax of 13.0 pmol/mg in human placental membranes. The adenosine A2-like binding site was purified after extraction from placental membranes with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The purification included ammonium sulfate precipitation and concanavalin A, DEAE-Sephadex, and Sepharose 6B gel filtration chromatographies. The protein was purified 127-fold to homogeneity, with a final specific activity of 1.5-1.9 nmol/mg of protein and a 5.5-8.1% yield of binding activity from the membranes. The purified protein had similar binding properties and an identical potency order for displacement of [3H] NECA by adenosine analogs as the initial membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa which coeluted with [3H]NECA binding activity during Sepharose 6B gel filtration chromatography. In 0.1% Triton X-100, the binding complex has a Stokes radius of 70 A, a sedimentation coefficient of 6.9 S, and a partial specific volume of 0.698 ml/g. The detergent-protein complex has a calculated molecular mass of 230 kDa. The estimated frictional ratio is 1.5. The native binding complex appears to consist of a dimer of identical subunits. The function of this ubiquitous protein remains unclear.     

Purification and properties of a beta-1,6-glucanase from Penicillium brefeldianum.

An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.     

Molecular cloning and characterization of an endo-1,3-beta-D-glucanase from the mollusk Spisula sachalinensis

cDNA encoding the endo-1,3-beta-d-glucanase from Spisula sachalinensis (LIV) was amplified by PCR using oligonucleotides deduced from the N-terminal end peptide sequence. Predicted enzyme structure consists of 444 amino acids with a signal sequence. The mature enzyme has 316 amino acids and its deduced amino acid sequence coincides completely with the N-terminal end (38 amino acids) of the beta-1,3-glucanase (LIV) isolated from the mollusk. The enzyme sequence from Val 121 to Met 441 reveals closest homology with Pacifastacus leniusculus lipopolysaccharide- and beta-1,3-glucan-binding protein and with coelomic cytolytic factors from Lumbricus terrestris. The mollusk glucanase also shows 36% identity and 56% similarity with beta-1,3-glucanase of the sea urchin Strongylocentrotus purpuratus. It is generally considered that invertebrate glucanase-like proteins containing the bacterial glucanase motif have evolved from an ancient beta-1,3-glucanase gene, but most of them lost their glucanase activity in the course of evolution and retained only the glucan-binding activity. A more detailed evaluation of the protein folding elicited very interesting relationships between the active site of LIV and other enzymes, which hydrolyze native glucans.     

Immunochemical characterization of the human blood cell membrane glycoprotein recognized by the monoclonal antibody 12E7.

The 12E7 murine monoclonal antibody recognizes a protease-sensitive component of human red cells, platelets and lymphocytes which could not be detected on granulocytes. Scatchard analyses indicated that the 125I-labelled antibody binds to 1000, 4000 and 27,000 antigen sites on each red cell, platelet and lymphocyte respectively, with a binding constant ranging from 4 x 10(7) to 9 x 10(7) M-1. The membrane components recognized by the monoclonal antibody were characterized by immunostaining on nitrocellulose sheets. A 28 kDa sialoglycoprotein was visualized following electrophoretic transfer of the red cell and lymphocyte membrane proteins separated by SDS/polyacrylamide-gel electrophoresis. Another component of 25 kDa was also clearly identified in the lymphocyte and platelet lysates, but was barely detectable in the red cell membrane preparations. Enzyme treatment of intact platelets, as well as analysis of the membrane and cytosolic preparations from these cells, have shown that the 25 kDa component was of cytoplasmic origin. The mobility of the 28 kDa membrane component is decreased following neuraminidase treatment of intact blood cells, but these cells still react normally with the monoclonal antibody, indicating that sialic acids are not required for binding. The 28 kDa component is present on red cell membranes prepared from S-s-U-, En(a-) and Gerbich(-) individuals, demonstrating that it is a new sialoglycoprotein not derived from glycophorins A, B, C or D. The 28 kDa component was totally solubilized with 0.1% Triton X-100 from red cell membranes and behaves like the other red cell membrane sialoglycoproteins since it was extracted in the aqueous phase following chloroform/methanol/water or butanol/water partitionings. The 28 kDa component could be partially purified by h.p.l.c. gel permeation chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The material finally obtained strongly inhibits the 12E7 monoclonal as well as human anti-Xga antibodies, suggesting either that the 28 kDa glycoprotein carries both antigens or that the 12E7 and Xga-active molecules copurified.     

Isolation and renaturation of alpha 2-macroglobulin receptor from diploid human fibroblasts.

alpha 2-Macroglobulin receptor was extracted from human diploid fibroblasts and purified by affinity chromatography in a single step. The receptor had mol.wt. 125 000 after sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. The isolated receptor was separated by SDS/polyacrylamide-gel electrophoresis, transferred on to nitrocellulose sheets and subsequently renatured, as shown by a specific binding test, by incubation with Nonidet P40.     

Studies on manganese binding by selective solubilization of Photosystem-II polypeptides

Deoxycholate was used to solubilize the 16 and 24 kDa polypeptides from spinach thylakoids, resulting in the loss of oxygen evolution. Manganese was retained in the membrane. When the deoxycholate-extracted membranes were subjected to a mild heat treatment, the water-soluble 33 kDa protein was selectively released. Less than one manganese per reaction center was lost on heating but this loss was not correlated to the solubilization of protein. Most of the manganese bound to the membrane remained EPR-undetectable and could be released by 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) or hydroxylamine treatments. This indicates that the manganese involved in oxygen evolution remains in its native binding site despite the loss of the 33 kDa protein. These results contradict the hypothesis that the 33 kDa protein is responsible for manganese binding at the photosynthetic oxygen-evolving site.     

Effect of deglycosylation on the structure and hormone-binding activity of the lutropin receptor.

Affinity-purified rat ovarian lutropin (LH) receptor is a single 90 kDa polypeptide which binds to immobilized lectins, indicating that the receptor is a glycoprotein [Keinänen, Kellokumpu, Metsikkö & Rajaniemi (1987) J. Biol. Chem. 262, 7920-7926]. In the present study the glycoprotein nature of the rat ovarian LH receptor was investigated in order to determine the contribution of the glycan moiety to receptor''s size and hormone-binding properties. Treatment of the 125I-labelled purified LH receptor with neuraminidase and peptide N-glycosidase F resulted in a decrease in size of LH receptor from 90 kDa to 79 kDa and 62 kDa respectively, as assessed by SDS/polyacrylamide-gel electrophoresis. Endo-alpha-N-acetylgalactosaminidase treatment did not affect the electrophoretic mobility of the intact or neuraminidase-treated LH receptor. Subjecting the membrane-bound LH receptor to similar enzymic treatments followed by ligand blotting showed that the 79 kDa and 62 kDa forms are capable of specific hormone binding. Furthermore, intact and peptide N-glycosidase F-treated membranes bound 125I-labelled human choriogonadotropin with similar affinities. These data suggest that molecular mass of the polypeptide backbone of the LH receptor is 62 kDa. The receptor contains N-glycosidically linked oligosaccharide chains with terminal sialic acid residues, with little or no O-linked oligosaccharide. N-Linked carbohydrate is not required for specific high-affinity hormone binding.     

Rat intestinal trehalase. Studies of the active site.

Rat intestinal trehalase was solubilized, purified and reconstituted into proteoliposomes. With octyl glucoside as the solubilizing detergent, the purified protein appeared as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 67 kDa. Kinetic studies indicated that the active site of this enzyme can be functionally divided into two adjacent regions, namely a binding site (with pKa 4.8) and a catalytic site (with pKa 7.2). Other findings suggested that the catalytic site contains a functional thiol group, which is sensitive to inhibition by N-ethylmaleimide, Hg2+ and iodoacetate. Substrate protection and iodoacetate labelling of the thiol group demonstrated that only a protein of 67 kDa was labelled. Furthermore, sucrose and phlorizin protected the thiol group, but Tris-like inhibitors did not. Structure-inhibition analysis of Tris-like inhibitors, the pH effect of Tris inhibition and Tris protection of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide inactivation permitted characterization and location of a separate site containing a carboxy group for Tris binding, which may also be the binding region. On the basis of these findings, a possible structure for the active site of trehalase is proposed.     

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors.     

Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.     

A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.     

Phosphoproteins and protein-kinase activity in isolated envelopes of pea (Pisum sativum L.) chloroplasts

A protein kinase was found in envelope membranes of purified pea (Pisum sativum L.) chloroplasts. Separation of the two envelope membranes showed that most of the enzyme activity was localized in the outer envelope. The kinase was activated by Mg²⁺ and inhibited by ADP and pyrophosphate. It showed no response to changes in pH in the physiological range (pH 7-8) or conventional protein substrates. Up to ten phosphorylated proteins could be detected in the envelope-membrane fraction. The molecular weights of these proteins, as determined by polyacrylamide-gel electrophoresis were: two proteins higher than 145 kDa, 97, 86, 62, 55, 46, 34 and 14 kDa. The 86-kDa band being the most pronounced. Experiments with separated inner and outer envelopes showed that most labeled proteins are also localized in the outer-envelope fraction. The results indicate a major function of the outer envelope in the communication between the chloroplast and the parent cell.     

Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete<Emphasis Type="Italic"> Mucor circinelloides</Emphasis>

We have produced the rat adenosine A1 receptor in Mucor circinelloides using a translational fusion to the endogenous glucoamylase (GlaM) gene. The fusion protein produced from an episomal plasmid was correctly processed as judged by western blotting, since only a 33 kDa band was detected in membrane preparations from M. circinelloides expressing the receptor. This corresponds to the mass of the full-length receptor released from the fused GlaM protein. The presence of a high affinity binding site with a K_d value of 0.5 nM for the receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in membrane preparations suggests that the receptor was correctly folded and inserted into the membranes. A receptor expression level of 100–300 fmol/mg total membrane protein was achieved as judged by binding of the antagonist [³H]-DPCPX.