Storage globulins in Gnetopsida. 2. A serotaxonomic study of legumin-like proteins

Abstract

Legumin-like proteins from Ephedra distachya, Ephedra foeminea, Gnetum gnemon, Gnetum montanum and Welwitschia mirabilis and four Angiosperms (Magnolia grandiflora, Pisum sativum, Glycine max and Cucurbita moschata) were used in a serological investigation. The immunological tests demonstrate the heterogeneity of Gnetopsida and indicate discriminative correlations of Welwitschia, Gnetum and Ephedra to the examined Angiosperms.     

The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds

Two proteolytic enzymes, a cysteine proteinase and a carboxypeptidase, responsible for breakdown of the main storage protein, 13S globulin, were purified from buckwheat seedlings (Fagopyrum esculentum Moench) by (NH₄)₂SO₄ fractionation, gel-filtration on Sephadex G-150, ionexchange chromatography on DEAE-Toyopearl 650 M and chromatofocusing. The cysteine proteinase was purified 74-fold. It has a pH optimum of 5.5, a pI of 4.5 and an apparent molecular mass (M_r) of 71000. The carboxypeptidase was purified 128-fold. It has a pH optimum of 5.3, a pI of 5.8 and a M_r of 78500. Cysteine proteinase hydrolyzed the modified 13S globulin only if the reaction products were eliminated from the incubation mixture by dialysis. Storage protein degradation by the proteinase increased in the presence of carboxypeptidase. We suggest that the two enzymes complete the digestion of 13S globulin after its preliminary hydrolysis by the earlier described enzyme, metalloproteinase, present in dry buckwheat seeds.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - M_r apparent molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Oat seed globulin: subunit characterization and demonstration of its synthesis as a precursor

The predominant storage protein of oat (Avena sativa L.) seeds is a saline-soluble globulin with a mol wt of 320,000 which is composed of six large (M_r = 35,000 to 40,000) and six small (M_r = 20,000 to 25,000) subunits. Experiments were conducted to further describe the subunit polypeptides and to identify the initial translation products of globulin mRNAs. Approximately 20 large subunits and 10 small subunits were resolved by two-dimensional gel analysis. The large and small subunits had acidic and basic isoelectric points, respectively. Disulfide-linked complexes of one large and one small subunit were isolated by extraction in buffer lacking a reducing agent. The NH₂-terminal sequence of the small subunits was homologous to a small subunit of soybean glycinin. Immunoprecipitation of in vitro translation products of poly(A)⁺ RNA with anti-oat globulin sera yielded M_r = 60,000 to 68,000 polypeptides. In vivo labeling of spikelets with radioactive amino acids resulted in high amounts of incorporation into polypeptides with M_r = 65,000 to 68,000 which were immunoprecipitated with anti-globulin sera. These two results suggest oat globulin is synthesized as a higher mol wt precursor which is subsequently processed to yield the large and small subunit polypeptides.     

Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs

Summary Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constitutent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins ofM _r 32 and 22 kD in rat and mouse liver, ofM _r 41 kD in rat cerebellum, ofM _r 43 kD in uterus, stomach and kidney of rat, ofM _r 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.     

β-1,3-glucanase and chitinase as pathogenesis-related proteins in the defense reaction of twoCapsicum annuum cultivars infected with cucumber mosaic virus

Pathogenesis-related (PR) proteins from pepper (Capsicum annuum L.) cv. Americano (tolerant) and cv. Smith-5 (sensitive), both elicited by infection with cucumber mosaic virus (CMV), were assayed for chitinase and glucanase activities. Two basic PR-proteins, M_r 49.0 and 28.0 kD, were elicicited from the intracellular fraction (INTRA-F) of both cvs. by CMV infection, while four acidic M_r 15, 19, 36 and 40 kD and two basic M_r 21.2 and 24 kD PR-proteins were elicited from the intercellular fluid (IF) of cv. Americano leaves. Five acidic M_r 21.5, 23.2, 24.4, 25.2 and 36 kD and five basic M_r 23.3, 26, 28.8, 30 and 32.3 kD PR proteins were elicited from the IF of cv. Smith-5. Isoelectric focusing (IEF) of the IF and the INTRA-F proteins revealed the occurrence, in both pepper cultivars, of one acidic M_r 36 kD and one basic M_r 25 kD PR-protein with glucanase activity. After native-PAGE for acidic proteins, the acidic PR-protein of Rf 0.7 and M_r 36 kD present in the IF of both pepper cvs. showed glucanase activity. Native-PAGE for basic proteins of the INTRA-F showed the presence of one band (Rf 0.61, M_r 25 kD) common to both cvs. and two additional bands (Rf 0.49, M_r 26 kD and Rf 0.79, M_r 33 kD) in the cv. Americano with glucanase activity. The specificity shown by the basic PR-proteins suggests glucanase activity is involved in the mechanisms of resistance to CMV in the cv. Americano. There was no difference in chitinase isoform patterns between the two pepper cultivars analyzed. After IEF of the IF proteins, one acidic chitinase isoform was detected. Native-PAGE separation of the IF showed one band (M_r 30 kD) with chitinase activity. Chitinase activity was not detected in the INTRA-F of either cultivar.     

Subunit composition of the globulin fraction of rapeseed (Brassica napus L.)

Multidimensional gel electrophoretic procedures have been employed to studythe polypeptide composition of the 12 S globulin (cruciferin) from rapeseed (Brassica napus L. cv. Tandem). Cruciferin was shown to consist of three main groups of proteins with M_r in the range of 60-52 (A group), 37-35 and 31-29 (respectively B₁ and B₂ groups) and 24-22 (C group). When reduced the A protein group gave rise to two classes of polypeptide constituents. The larger had M_r and isoelectric points (6.4–7.1) corresponding to those of B protein groups whereas the smaller were essentially composed of M_r 22 and a specific 25 kilodaltons (kD) polypeptide with basic isoelectric points. The initial B and C protein groups were unaltered by reducing conditions. These results support the notion that native cruciferin is composed of subunits with large and small polypeptides linked by disulphide bonds and of similar or closely-related polypeptides which are not covalently bonded.     

The high-molecular-weight proteins of bovine brain plain synaptic vesicles

High molecular mass polypeptides (M _r >100,000) of plain synaptic vesicles from bovine cerebral cortex were separated using porous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Four major bands, ofM _r 262,000, 249,000, 216,000, and 173,000, were resolved. Investigations into the membrane association of theM _r 216,000 and 173,000 proteins by means of solubilization experiments and Sepharose 4B chromatography indicate that the former is a peripheral protein and the latter is more firmly attached, possibly an integral protein. Finally, theM _r 216,000 protein was shown to be highly enriched in synaptic vesicles compared to other brain subfractions. It thus appears to be specifically associated with synaptic vesicles and therefore may have an important role specific to synaptic vesicle function or structure.     

Identification of Several Pathogenesis-Related Proteins in Tomato Leaves Inoculated with Cladosporium fulvum (syn. Fulvia fulva) as 1,3-beta-Glucanases and Chitinases

Inoculation of tomato (Lycopersicon esculentum) leaves with Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) results in a marked accumulation of several pathogenesis-related (PR) proteins in the apoplast. Two predominant PR proteins were purified from apoplastic fluid by ion exchange chromatography followed by chromatofocusing. One protein (molecular mass [M_r] 35 kilodaltons [kD], isoelectric point [pI] ~6.4) showed 1,3-β-glucanase activity, while the other one (M_r26 kD, pI ~6.1) showed chitinase activity. Identification of the products that were released upon incubation of the purified enzymes with laminarin or regenerated chitin revealed that both enzymes showed endo-activity. Using antisera raised against these purified enzymes from tomato and against chitinases and 1,3-β-glucanases isolated from other plant species, one additional 1,3-β-glucanase (M_r33 kD) and three additional chitinases (M_r 27, 30, and 32 kD) could be detected in apoplastic fluids or homogenates of tomato leaves inoculated with C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-β-glucanase activity in apoplastic fluids increased more rapidly in incompatible interactions than in compatible ones. The role of these hydrolytic enzymes, potentially capable of degrading hyphal walls of C. fulvum, is discussed in relation to active plant defense.     

Enzyme antigens associated with pigeon breeder's disease. I. Isolation and characterization of basic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (M _r =22,000). The purified trypsin had a molecular weight (M _r =25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (M _r =51,000) was shown to be a glycoprotein consisting of two polypeptides (M _r =24,000). It was readily separated from the low-molecular-weight collagenase (M _r =15,000) by gel filtration. The phospholipase (M _r =99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.     

Catfish Thrombocytes Express an Integrin-like CD41/CD61 Complex

A thrombocyte-specific antigen was identified in two closely related catfish,Ictalurus punctatusandIctalurus furcatus,by monoclonal antibodies 4-20 and 7-2. The antibodies immunoprecipitate two noncovalently associated glycoprotein chains ofM_r180,000 andM_r95,000. Under reducing conditions theM_r180,000 chain is resolved intoM_r150,000 and 32,000 subcomponents. Analysis of N-terminal amino acid sequences indicates homology of theM_r95,000 chain with the β₃integrin subunit and homology of theM_r150,000 chain with the α_IIbintegrin subunit. These antibodies induce catfish thrombocyte aggregation and alteration of cell shape. The data indicate conservation of the megakaryocyte/platelet-restricted CD41/CD61 complex in bony fish.     

Biotinylation of proteins on the surface of zona-free mouse oocytes

In this study, we have labelled proteins on the surface of unfertilized, zona-free mouse oocytes using a nonisotopic biotinylation procedure. The zona pellucida was weakened by brief incubation in chymotrypsin and removed by mechanical pipetting through a narrow-bore glass pipette. Surface proteins were labelled using sulfo-NHS-biotin (sulfosuccinimidobiotin), a water-soluble, membrane-impermeable biotinylation reagent. The distribution of biotinylated proteins on the oocyte surface was assessed by fluorescence microscopy using streptavidin–FITC. Bright fluorescence was noted on the surface of the oocyte, except in a circular region overlying the meiotic spindle where the fluorescence was weak or absent. The intensity of fluorescence was markedly reduced by incubation of biotinylated oocytes in trypsin (1 mg/ml) or chymotrypsin (2 mg/ml), and in vitro fertilization experiments showed that biotinylation did not compromise the fertilizability of the oocytes. The biotinylated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by Western blotting using streptavidin–HRP and enhanced chemiluminescence (ECL) detection. The most prominent biotinylated proteins were of M_r 82 and 69 kD, but other major proteins of M_r 93, 78, 61, 52, 49, 40, 28, and 22 kD were detected, as well as 14 minor proteins of M_r 18–100 kD. The major bands could be detected in fewer than 50 oocytes. This biotinylation procedure is fast, versatile and sensitive, and it is therefore an excellent tool for studying proteins exposed on the surface of mammalian oocytes and embryos. © 1993 Wiley-Liss, Inc.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Rat epididymis-specific sperm maturation antigens. I. Evidence that the 26 kD 4E9 antigen found on rat caudal epididymal sperm tail is derived from a protein secreted by the epididymis

Monoclonal antibody 4E9, which was raised against a partially purified detergent extract of rat caudal epididymal sperm, recognizes the tail of sperm from the cauda, but not from caput epididymidis, as well as epithelial cells in a restricted region of the distal caput/corpus epididymidis and proteins in epididymal fluid from corpus and cauda epididymidis. The antigen is apparently a glycoprotein, since it is retained on a Ricinus communis agglutinin l lectin column. Epididymal fluid antigens have apparent M_rS of 38–26 kD, whereas the memrane-associated form of the molecule has an M_r of 26 kD. Immunocytochemical data and Western immunoblot data suggest that the membrane antigen is derived from the fluid antigen, which, in turn, is secrteted by the epididymal epithelium. Characterization of the membrane antigen indicates that it is tightly associated with the sperm surface, behaving as though it is an integral membrane protein. The antigen persists on ejaculated sperm. © 1994 Wiley-Liss, Inc.     

Etude de la globuline des graines de Tournesol (Helianthus annuus L.) accumulation au cours de la maturation et localisation intracellulaire

Protein synthesis and accumulation in growing sunflower (Helianthus annuus L.cv.Airelle) seeds are studied. The salt soluble fraction, globulin, is the main soluble protein component. The earlier stages of seed development (10 days after flowering) are characterized by high M_r polypeptides (74, 58 and 44 kDa). Later stages mainly show nature globulin polypeptides. Thus, protein synthesis in seed occurs at a specific period of seed development which follows a period of fast cell divisions (0–14 days after flowering). Protein bodies are isolated and their protein composition analyzed. Globulin subunits are the main polypeptides of protein bodies soluble fraction. Mature globulin is only stored in protein bodies.      

Purification and characterization of three major hemolymph proteins of an insect,Calpodes ethlius (lepidoptera,hesperiidae)

Like many other Lepidoptera, fifth-stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3-15% nondenaturing polyacrylamide gel electrophoresis (N-PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N-PAGE and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On 3-15% SDS gels, Ar dissociated into 82,000 M_r subunits, SP2 into 86,000 M_r subunits, and SP1 into both 86,000 and 90,000 M_r subunits. The 470,000 M_r protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 M_r proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.     

C-GLYCOSYLFLAVONES IN THE GNETOPSIDA: A PRELIMINARY REPORT

C-Glycosylflavones appear to be the only detectable flavonoids in representative species of Gnetum, Ephedra and Welwitschia. Ephedra antisyphilitica contains mixed 6,8-di-C-glycosides of apigenin and luteolin and Welwitschia mirabilis produces 6-C-glycosylchrysoeriol and 6,8-di-C-glycosylchrysoeriol. Recently it was reported that Gnetum gnemon contains 6-C-, 8-C and 6,8-di-C-glucosyl derivatives of apigenin, luteolin and their 7-0-methyl ethers. Based on the present preliminary survey the apparent absence of other flavonoid types indicates that C-glycosylflavones may be a unifying characteristic of the Gnetopsida.     

Enhanced fibroblast collagen production by a macrophage-derived factor (CEMF)

A basic fraction with pl values of 10.0–10.4 was isolated from macrophage culture medium. This fraction stimulated the production of collagen into fibroblast culture medium but inhibited the production of other proteins. Both changes were strongly dependent on the concentration of the factor. As revealed by SDS-PAGE the production of type I collagen was especially stimulated. The specific enhancement of fibroblast collagen production was also observed in the presence of serum proteins. The collagen synthesis enhancing macrophage-derived factor preparation (CEMF) contained three proteins, one major (M_r 23 kD) and two minor (M_r 49 and 71 kD) fractions in SDS-PAGE. When prelabeled fibroblast medium was exposed to CEMF a protein with M_r of 350 kD or greater was converted to a smaller size. This change was inhibited by EDTA but not by serum proteinase inhibitors.     

Purification and Properties of Methanol Dehydrogenase and Aldehyde Dehydrogenase from Methylobacillus glycogenes

Methanol dehydrogenase (MDH) and aldehyde dehydrogenase (ALDH) were purified to a homogenous state from Methylobacillus glycogenes, an obligate methylotroph. MDH (M_r 140,000) was composed of two different subunits (M_r 60,000 and 9,000) forming an α₂β₂ structure. MDH was indicated as a metalloquinoprotein containing one atom of calcium (Ca) per enzyme molecule. Binding of Ca was so tight that it was hard to remove Ca completely without denaturation of enzyme protein. A partially resolved enzyme resumed its original enzyme activity upon exogenous addition of Ca. Purified ALDH (M_r 144,000) was composed of two identical subunits of molecular mass of 72,000. ALDH was proved to be a quinoprotein in which PQQ is bound covalently.     

Proteolysis of cardiac gap junctions during their isolation from rat hearts

Summary Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a M_r 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a M_r 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol. 246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a M_r 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the M_r 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the M_r 29,500 subunit. We show that the M_r 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition.     

Polypeptide composition and biosynthesis of the yolk proteins in the firebrat,Thermobia domestica (insecta,thysanura)

Ion-exchange chromatography of crude ovarian extracts of the primitive insect Thermobia domestica allowed the separation, in native conditions, of major and minor vitellins of molecular weights of 300,000 and 430,000, respectively. Their polypeptide subunits were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunotransfer using an antiserum prepared against major vitellin. This protein was resolved into large (M_r 166,000–212,000) and small (around M_r 50,000) polypeptides. Minor vitellin, on the other hand, exclusively contained small polypeptides that are immunologically different from those of the major vitellin. Vitellogenin polypeptides from the hemolymph of mature females exhibited electrophoretic mobilities and immunological properties similar to vitellin polypeptides. Pulse-chase experiments showed that the female fat body synthesizes radioactive and immunoprecipitable proteins, whose polypeptide pattern is close to that of the major vitellogenin. However, part of the primary vitellogenic polypeptides, at M_r 210,000 and 212,000, is rapidly processed to M_r 176,000 and 182,000 subunits. These two polypeptides, as well as the precursors, enter into the composition of the major hemolymph vitellogenin. Finally, processing of the still uncleaved 210,000–212,000 polypeptides takes place in the ovary, which performs the same step of vitellogenin maturation as the fat body.