Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.     

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.     

Molecular Species in the Protein Body II (PB-II) of Developing Rice Endosperm

The molecular weight and subunit composition of glutelin, the major storage protein of rice, in the major type of protein bodies of developing rice seeds was examined by gradient and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Glutelin in the protein body was the assembled from heterogeneous subunits, and the molecular weights were estimated to be 64, 140, 240, 320, 380, and 500 k by gradient SDS-PAGE. High molecular weight proteins (larger than 2,000 k) were also observed.

The two-dimensional SDS-PAGE under reduced conditions showed, that the glutelin in the protein body was composed of two groups of polypeptides, 22~23 and 37~39k, bound by disulfide linkages.     

Sugar uptake by cotton tissues: leaf disc versus cultured roots

The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.

Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The α and β globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The α and β subunits appeared during the same chase period and assembled into a 12S oligomer.

The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the α and β subunits forming a 12S oligomer.

     

A Complex Array of Proteins Related to the Multimeric Leucine Aminopeptidase of Tomato

Leucine aminopeptidase (LAP) mRNAs are induced in response to mechanical wounding, pathogen infection, and insect infestation (V. Pautot, F.M. Holzer, B. Reisch, L.L. Walling [1993] Proc Natl Acad Sci USA 90: 9906-9910). Polyclonal antibodies to a glutathione S-transferase-LAP fusion protein and affinity-purified antibodies recognizing LAP antigenic determinants detected four classes of polypeptides in tomato (Lycopersicon esculentum) leaves. All four classes had multiple polypeptides in two-dimensional polyacrylamide gel electrophoresis immunoblots. Although antigenically related to the wound-induced tomato LAP proteins, the 77- and 66-kD LAP-like proteins accumulated in both healthy and wounded leaves. Two classes of 55-kD polypeptides with distinctive isoelectric points were designated as plant LAPs; only the acidic LAP proteins accumulated to high levels after mechanical wounding or Pseudomonas syringae pv tomato infection of tomato leaves. The temporal accumulation of LAP mRNAs was correlated with the increase in acidic LAP protein subunits. A slow-migrating LAP activity was detected using a native gel assay after wounding. The molecular mass of the native wound-induced LAP enzyme was 353 kD. The 55-kD acidic LAP proteins were associated with induced LAP activity, whereas the neutral LAPs and the LAP-like proteins were not associated with this exopeptidase. A second, fast-migrating aminopeptidase was detected in both healthy and wounded tomato leaves. Cell fractionation experiments revealed that wound-induced LAP is a soluble enzyme.     

蛋白酶抑制剂对含水生菜种子耐冻性的影响

选取生菜(Lactuca sativa)种子作为试材,外源添加蛋白酶抑制剂2-硝基苯甲酸[5,5'-Dithiobis-(2-nitrobenzoic acid),DTNB]对种子吸胀处理,通过程序降温,分析2-硝基苯甲酸对低温下种子发芽率及生理活性的影响。结果表明,低温下含水生菜种子的致死温度为–20 ℃;外源添加2-硝基苯甲酸2 mmol·L–1时种子发芽率最高,即对种子活性的保护效果显著;在此浓度下种子内SOD活性比对照提高1.38倍,羟基自由基清除能力提高1.17倍;与对照组相比产生两种新的蛋白11 S种子贮藏球蛋白Jug r4和11 S种子贮藏球蛋白2,均属于球蛋白家族,可提高含水种子的耐冻性;低温下种子内积累更小分子量的球蛋白多肽,对种子具有低温保护效果。综上,低温条件下生菜种子产生一定的抗冷反应,外源添加2 mmol·L–1 2-硝基苯甲酸可提高含水种子发芽率及生理活性,产生抗冻蛋白,积累更小分子量的球蛋白多肽进而提高种子抗冻性。     

Separation and characterization of oat globulin polypeptides

The storage globulin of oat seeds was separated into its acidic (α) and basic (β) polypeptides by ion-exchange chromatography in 6 m urea and further characterized by several electrophoretic techniques. Molecular weights of the α and β polypeptides were 32,500–37,500 and 22,000–24,000, respectively. The unreduced protein existed as disulfide-linked αβ species of molecular weight 53,000–58,000. Isoelectric points were approximately 5.9–7.2 (α) and 8.7–9.2 (β). Two-dimensional electrophoresis showed considerable heterogeneity within both groups of polypeptides. More complete amino acid analyses of the globulin and its polypeptides are presented along with a proposed structure of the native protein based on previous and present data. Similarities were noted between the oat globulin and the legumin (11 S) class of storage proteins in certain legumes.     

Analysis of globulin maturation in developing sunflower seeds

The synthesis of major storage globulin polypeptides has been examined in developing seeds of sunflower(Helianthus annuus L.). Analyses of total proteins and purified globulins, also called helianthinin, by gel electrophoresis and immunoelectrophoresis have shown that a burst of protein synthesis and accumulation occurs around 10 d after flowering. There is no mature globulin before that time and only small amounts of precursor forms can be detected. Thus, 10–12 d after flowering appears to be a transition period during which genetic information for the globulin becomes actively expressed. Immunoelectrophoresis has confirmed that globulin is the main storage protein, at seed maturation, accounting up to 70 % of total proteins per kernel. Pulse chase experiments have shown that synthesis initially involves the formation of high molecular mass precursors and that storage proteins are post-translationally processed. Intermediary products, with molecular mass higher than early translational products, can be detected, together with mature globulin polypeptides.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco

Zeins, the seed storage proteins of maize, are a group of alcohol-soluble polypeptides of different molecular masses that share a similar amino acid composition but vary in their sulfur amino acid composition. They are synthesized on the rough endoplasmic reticulum (ER) in the endosperm and are stored in ER-derived protein bodies. Our goal is to balance the amino acid composition of the methionine-deficient forage legumes by expressing the sulfur amino acid-rich 15-kD zeins in their leaves. However, it is crucial to know whether this protein would be stable in nonseed tissues of transgenic plants. The major focus of this paper is to compare the accumulation pattern of the 15-kD zein protein with a vacuolar targeted seed protein, [beta]-phaseolin, in nonseed tissues and to determine the basis for its stability/instability. We have introduced the 15-kD zein and bean [beta]-phaseolin-coding sequences behind the 35S cauliflower mosaic virus promoter into tobacco (Nicotiana tabacum) and analyzed the protein's accumulation pattern in different tissues. Our results demonstrate that the 15-kD seed protein is stable not only in seeds but in all nonseed tissues tested, whereas the [beta]-phaseolin protein accumulated only in mid- and postmaturation seeds. Interestingly, zein accumulates in novel protein bodies both in the seeds and in nonseed tissues. We attribute the instability of the [beta]-phaseolin protein in nonseed tissues to the fact that it is targeted to protease-rich vacuoles. The stability of the 15-kD zein could be attributed to its retention in the ER or to the protease-resistant nature of the protein.     

Studies on proteins that co-purify with smooth muscle vinculin: identification of immunologically related species in focal adhesions of nonmuscle and Z-lines of muscle cells

Membrane extracts from chicken smooth muscle contain, along with filamin, vinculin and alpha actinin, a group of polypeptides that have the ability to interact with the "barbed end" of actin filaments. These low molecular mass polypeptides were designated as HA1 (Wilkins, J.A., and S. Lin, 1986, J. Cell Biol., 102:1085-1092). In this study, polyclonal antibodies raised against the HA1 preparation were used to study the cellular localization and tissue distribution of these polypeptides. Immunofluorescence experiments revealed a primary localization of staining at the ends of stress fibers on the ventral surface of cultured chicken embryo fibroblasts, i.e., those areas known as the focal adhesions. Specific staining was also seen at the Z-lines of both skeletal muscle myofibrils and cultured embryonic heart cells. Immunoblotting analyses of proteins from different tissues prepared to avoid proteolytic degradation showed a much different pattern than that of HA1 itself. Immunoreactive polypeptides with reduced molecular masses of 200,000 and 150,000 D were found in smooth muscle and fibroblasts while 200 and 60 kD polypeptides were found in cardiac muscle tissue. The antibodies recognized 60- and 31-kD polypeptides on immunoblots of chicken breast muscle. The results from this study strongly suggest that the polypeptides in HA1 arose from proteolysis of high molecular mass molecules. The studies also raise the possibility that immunologically related proteins in muscle and nonmuscle cells may be involved in linking actin filaments to Z-lines and membranes, respectively.     

Immunolocalization of avenin and globulin storage proteins in developing endosperm of Avena sativa L.

The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.Abbreviations DAA days after anthesis - IgG immunoglobulin G - M_r apparent molecular mass - RER rough endoplasmic reticulum - SDS-PAGE sodium dodecyl sulfate — polyacrylamide gel electrophoresis     

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.      

Proteolytic Cleavage at Twin Arginine Residues Affects Structural and Functional Transitions of Lupin Seed 11S Storage Globulin

The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.     

Identification of the 7S globulin with β-conglycinin in soybean seeds

The 7S globulin, a major ultracentrifugal component with the 11S globulin, was identical with β-conglycinin one of four antigenic components in the reserve proteins of soybean seeds (Glycine max). Double gel immunodiffusion and immunoelectrophoresis in agar gel were used for their identification. In addition, some characteristic properties on ultracentrifugation and in carbohydrate content agreed well between the proteins. Their MWs were ca 180000.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Structural Proteins and Subunits of Rubella Virus

Polyacrylamide gel electrophoresis of purified rubella virus revealed two distinct structural proteins VP1 and VP3, which had molecular weights of 62,500 and 35,000, respectively. In addition, a broad variable peak, designated VP2, with a molecular weight of about 47,500, was seen. Sucrose gradient analysis of virus disrupted by neutral detergents separated a labile 150S ribonucleoprotein, containing 40S ribonucleic acid and VP3, from the envelope fraction containing VP1 and VP2. VP1 and particularly VP2 were labeled with glucosamine and are thus glycoproteins. Labeling the polypeptides with different amino acids indicated that VP3, the "core" protein, is relatively rich in arginine but not in lysine. The size of the two main polypeptides, VP1 and VP3, corresponds to those of group A arboviruses.     

The phosphorylation of coated membrane proteins in intact neurons

To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two-dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non-equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have previously described, as well as a novel 155-kD polypeptide reported here, have structural and functional counterparts in cultured neurons. They also indicate that phosphorylation of the 100-110-kD AP may be involved in the regulation of coated membrane structure and function. The extent of phosphorylation of the AP50 in intact cells and in isolated coated vesicles is strikingly different: it has been suggested that the latter process reflects an autophosphorylation reaction (Campbell C., J. Squicciarini, M. Shia, P. F. Pilch, and R. E. Fine, 1984, Biochemistry, 23:4420-4426).(ABSTRACT TRUNCATED AT 400 WORDS)     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

Sensitive Immunoassay Shows Selective Association of Peripheral and Integral Membrane Proteins of the Inhibitory Glycine Receptor Complex

The inhibitory glycine receptor of mammalian spinal cord is a ligand-gated chloride channel that, on affinity purification, contains two subunits of 48-kilodalton (kD) and 58-kD molecular mass in addition to an associated 93-kD protein. Ligand-binding 48-kD subunit and 93-kD protein were quantified in the CNS of the adult rat using a newly developed dot receptor assay (detection limit less than or equal to 1 fmol/assay) which employs monoclonal antibodies specific for glycine receptor polypeptides. The 93-kD protein was found to codistribute at a fixed stoichiometry with the 48-kD subunit throughout the CNS of the rat. Moreover, the 93-kD protein cofractionated with the ligand-binding subunit on solubilization and affinity chromatography or immunoprecipitation. However, both proteins were separated on sucrose gradient centrifugation of detergent extracts of spinal cord membranes in accord with earlier observations on purified receptor. These data prove that the 93-kD polypeptide is selectively associated with the membrane core of the strychnine-sensitive glycine receptor. The regional distribution of glycine receptor polypeptides was also determined in the CNS of the spastic rat mutant. In contrast to hereditary spasticity in mouse and cattle, no reduction of glycine receptors was found in the spastic rat.