The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.     

The N-terminal regions of beta and gamma subunits lower the solubility of adenosylcobalamin-dependent diol dehydratase

Adenosylcobalamin-dependent diol dehydratase is one of essential components of carboxysome-like polyhedral bodies. It exists as a heterohexamer (alphabetagamma)(2), and its activity is recovered in a precipitant fraction of Klebsiella oxytoca and overexpressing Escherichia coli cells. Limited proteolysis of the enzyme with trypsin converted the enzyme into a highly soluble form without loss of enzyme activity. The N-terminal amino acid sequencing of the enzyme thus solubilized indicated that the N-terminal 20 and 16 amino acid residues had been removed from the beta and gamma subunits, respectively. Mutant enzymes with the same N-terminal truncations of either or both of the beta and gamma subunits were expressed on a high level in E. coli cells. All the mutant enzymes obtained were expressed in a soluble, active form. These results indicate that the N-terminal regions of the beta and gamma subunits lower the solubility of diol dehydratase. The mutant enzyme with the N-terminal truncations of both beta and gamma subunits was essentially indistinguishable in catalytic properties from recombinant wild-type enzyme or the enzyme purified from K. oxytoca in a soluble form.     

One of two subunits of masking protein in latent TGF-beta is a part of pro-TGF-beta

A high molecular mass latent form of transforming growth factor type-beta (TGF-beta) was purified to homogeneity from rat platelets by a seven-step procedure involving group-specific affinity chromatographies on Red-Toyopearl and zinc chelating-Sepharose. The purified latent TGF-beta was a complex of TGF-beta (25 kDa) and the binding protein previously named masking protein (approximately 400 kDa) [(1986) Biochem. Biophys. Res. Commun. 141, 176-184]. Analysis of the peptide structure by gel electrophoresis showed that the masking protein consisted of two subunits of 39 kDa and 105-120 kDa linked by disulfide bonds. N-terminal amino-acid sequencing of the 39 kDa subunit indicated that this subunit was identical to the N-terminal part of the TGF-beta precursor.     

Structural Relationship among the Rice Glutelin Polypeptides

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the α and β subunits are proteolytic products of the 51 kD precursor protein. The poly(A)⁺ RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.     

Development of the cotyledon cells during olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) in vitro seed germination and seedling growth

The structural changes occurred in differentiating olive cotyledon cells into mesophyll cells are described. Using histological and immunocytological methods as well as microscopic observations, we showed that in the cells of mature embryo, large electron-dense proteins bodies (PBs) are surrounded by numerous oil bodies (OBs). After 3 days of in vitro germination, the presence of large PBs originated by fusion of smaller PBs was observed. It was also detected a close spatial proximity between PBs and OBs, likely as a reflection of interconnected metabolic pathways. Between the 3rd and the 12th day of germination, the formation of a large vacuolar compartment takes place accompanied by a decrease in the PBs and OBs number. This was coincident with a progressive decrease in the amount of the 11S-type seed storage proteins (SSPs), showed in situ and after Western blot analysis of crude protein extracts. After 26 days germination, the cellular organization became typical for a leaf mesophyll cell, with well-differentiated chloroplasts surrounding a large central vacuole. Our results suggest that the olive cotyledon storage reserves are mobilized gradually until the seedling becomes autotrophic. Moreover, the specific accumulation of storage proteins in the intravacuolar material suggests that these structures may operate as a shuttle for SSPs and/or products of their degradation into the cytoplasm, where finally they supply amino acids for the differentiating mesophyll cells.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

7S globulins from Phaseolus vulgaris L.: Impact of structural aspects on the nutritional quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS-PAGE showed major bands corresponding to the phaseolin subunits (43-53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 degrees C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of beta-sheet and beta-turn structures, together with a contribution at 1687 cm(-1) that was assigned to intramolecular beta-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.     

Extended N-terminal sequencing of proteins of archaebacterial ribosomes blotted from two-dimensional gels onto glass fiber and poly(vinylidene difluoride) membrane

Previously uncharacterized proteins from intact ribosomes and ribosomal subunits of the extreme halophile Halobacterium marismortui (Haloarcula marismortui) were isolated and separated by high-resolution two-dimensional electrophoresis (2DE). N-Terminal amino acid sequences of 14 of these acidic large-subunit proteins were obtained by direct blotting of the separated proteins from two-dimensional electrophoresis gels to sequencer-stable supports followed by excision of the protein spots and sequencing. Furthermore, long internal sequences were obtained by in situ enzymatic cleavage of halobacterial proteins in gel pieces obtained from two-dimensional gels followed by electrophoretic separation of the fragments, blotting, and sequencing. Precautions are outlined for avoidance of N-terminal blockage of proteins, and the preparation and selection of suitable supports for obtaining extended N-terminal sequences are described. The results suggest that when prior fractionation is carried out to enrich for cell organelles, subcellular components of cells, or cell membranes, it is routinely possible to obtain numerous N-terminal sequences from one or a few 2DE gels of such fractions. Our results also indicate that, with appropriate precautions, proteins are routinely obtainable from 2DE gels in a form suitable for both N-terminal and internal sequence determination and show no detectable evidence for N-terminal blockage or destruction or modification of labile amino acid residues.     

7S Globulins from Phaseolus vulgaris L.: Impact of Structural Aspects on the Nutritional Quality

7S globulins were extracted from common bean (Phaseolus vulgaris L.) seeds and characterized. SDS–PAGE showed major bands corresponding to the phaseolin subunits (43–53 kDa). An amino acid analysis indicated that, in spite of the limited amounts of sulphur amino acids and tryptophan, the globulins contained very high levels of essential amino acids. The protein solubility profiles of native and denatured (120 °C for 20 min) 7S globulins in water and in 0.5 M NaCl showed that NaCl had a limited effect on increasing the solubility of either the native or denatured proteins. The in vivo small intestinal digestibility of the 7S globulins was 90%, this being decreased to 86% after a thermal treatment. Fourier transform infrared spectroscopy revealed a high content of β-sheet and β-turn structures, together with a contribution at 1687 cm^?1 that was assigned to intramolecular β-sheets. These features are diagnostic of a high propensity to irreversible aggregation that may be related to an adverse effect on the protein quality.     

Relationships among the subunits of the high molecular weight proteinase,macropain (proteasome)

An analysis of the subunits of the high molecular weight proteinase, macropain (multicatalytic proteinase or proteasome) from human erythrocytes has been conducted using N-terminal amino acid sequencing, gel electrophoresis and reverse-phase peptide mapping. This analysis provided evidence for the existence of 13 subunits of different primary structure. Five subunits were susceptible to the Edman degradation and yielded unique N-terminal sequences. Similarities among these sequences, however, indicated that the subunits are homologues. Two-dimensional gel electrophoresis discriminated 10 major components, which included two of the subunits for which N-terminal sequences had been determined and eight N-terminally blocked subunits. Tryptic peptide mapping indicated that all 10 of these components have a different amino acid sequence. Tryptic peptides from some of the subunits were subjected to amino acid sequence analysis, and the data indicated that all the subunits tested in this way are related by common ancestry. The data suggest that at least nine of the total of 13 subunits are encoded by members of the same gene family; the remaining four subunits have not yet been investigated in sufficient detail to establish their relationships. No evidence for a close relationship with any previously investigated proteinase family has been found. Finally, through a comparison of the ‘latent’ and ‘active’ forms of macropain, the study established a close similarity in the subunit composition of these catalytically very different species, although proteolytic degradation of selected subunits appears in the active form of the enzyme.     

Formation of Pseudo- and Hybrid-11S Globulins from Subunits of Soybean and Broad Bean 11S Globulins

Pseudo- and hybrid-11S globulins were reconstituted from native acidic and basic subunits of soybean and broad bean 11S globulins. The subunit structures of these two globulins are known to be similar to each other. Pseudo-11S globulins were formed in combinations between glycinin acidic subunit (G-AS_{1 + 2}) and glycinin basic subunit (G-BS) and between legumin acidic subunit (L-ASII) and legumin basic subunit (L-BS). Hybrid-11S globulins were formed in combinations between G-AS_{1 + 2} and L-BS and between L-ASII and G-BS. The yields of the reconstituted 11S components of G-AS_{1 +2} + G-BS and G-AS_{1 + 2} + L-BS were lower than those of L-ASII + G-BS and L-ASII + L-BS. These pseudo- and hybrid-11S globulins were similar to native 11S globulins; they all consisted of reconstituted intermediary subunits which were composed of acidic and basic subunits linked by disufide bridges and had molecular weights similar to those of native 11S globulins. However, the dissociation-association behaviors of pseudo-glycinin and hybrid-11S globulins were different from those of native 11S globulins.     

Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20

The translocase of the outer mitochondrial membrane (TOM) complex is a preprotein translocase that mediates transport of nuclear-encoded mitochondrial proteins across the outer mitochondrial membrane. Here we report the purification of this protein complex from Arabidopsis. On blue-native gels the Arabidopsis TOM complex runs at 230 kD and can be dissected into subunits of 34, 23, 21, 8, 7, and 6 kD. The identity of four subunits could be determined by immunoblotting and/or direct protein sequencing. The 21- and the 23-kD subunits exhibit significant sequence homology to the TOM20 preprotein receptor from other organisms. Analysis by two-dimensional isoelectric focusing/Tricine sodium dodecyl sulfide-polyacrylamide gel electrophoresis revealed the presence of further forms for Arabidopsis TOM20. All TOM20 proteins comprise a large cytoplasmically exposed hydrophilic domain, which is degraded upon trypsination of intact mitochondria. Clones encoding four different forms of Arabidopsis TOM20 were identified and sequenced. The deduced amino acid sequences are rather conserved in the N-terminal half and in the very C-terminal part, but include a highly variable glycine-rich region close to the C terminus. Implications on the function of plant TOM complexes are discussed. Based on peptide and nucleic acid sequence data, the primary structure for Arabidopsis TOM40 is presented.     

Genetic characterization of Bordetella pertussis filamentous haemagglutinin: a protein processed from an unusually large precursor

The nucleotide sequence of the structural gene for filamentous haemagglutinin (FHA), fhaB, a crucial adherence factor for Bordetella pertussis, has been determined. Its 10774 nucleotides are far more than necessary to encode the 220 kD biologically active, mature polypeptide product, suggesting a role for co- or post-translational processing. Fusion proteins derived from various portions of the fhaB open reading frame (ORF) were used to generate polyclonal antisera. Western immunoblot analysis of purified FHA and Bordetella sp. whole cell extracts with these antisera indicated that the 220 kD product is encoded by the 5' portion of the ORF and that the smaller polypeptide species are breakdown products of this polypeptide. These data, as well as N-terminal amino acid sequencing of the major polypeptide species, suggest a scheme for the proteolytic processing of an FHA precursor polypeptide.     

莲子贮存蛋白的主要亚基及积累模式

红莲（NelumbonuciferaGaertn）成熟于叶总蛋白含量达24．35g／100g干样品,其贮存蛋白（SP）的积累模式与豆科种子相似,即随成熟度的提高,SP猛增至占总蛋白含量的86％以上。对莲子不同发育阶段子叶蛋白SDS-PAGE图谱的光密度测定表明：莲子叶蛋白有12个主要SP亚基（SP1－12）。按分子量（MW）大小和积累顺序可分3组：A组是98kD和93kD的2个亚基,MW最大,积累最晚,但量最多;B组是3个亚基,MW为55－50kD,大小居中,积累最早,量最少;C组的7个亚基MW最小,在27－14kD之间,积累较早,量较多。对莲的不同品种、不同器官进行分析后发现,它们都有1个主峰为68kD的多连峰的代谢蛋白亚基,可能是莲共有的蛋白亚基。     

Role of Auxin in Maize Endosperm Development (Timing of Nuclear DNA Endoreduplication,Zein Expression,and Cytokinin)

The timing of developmental events and regulatory roles of auxin were examined in maize (Zea mays L.) endosperms. Zeatin, zeatin riboside, and indole-3-acetic acid (IAA) levels were determined by enzyme-linked immunosorbent (ELISA). Zeatin and zeatin riboside increased to maximal concentrations at an early stage (9 d after pollination [DAP]), corresponding to the stage when cell division rate was maximal. In contrast, IAA concentration was low at 9 DAP and abruptly increased from 9 to 11 DAP, thus creating a sharp decline in the cytokinin to auxin ratio. Coincident with the increase in IAA was an increase in DNA content per nucleus, attributed to postmitotic DNA replication via endoreduplication. Exogenous application of 2,4-dichlorophenoxyacetic acid (2,4-D) at 5 or 7 DAP hastened the time course of DNA accumulation per nucleus and increased the average nuclear diameter, whereas 2-(para-chlorophenoxy)isobutyric acid delayed such development. Exogenously applied 2,4-D hastened the accumulation of the zein polypeptides of apparent molecular masses of 12, 14, and 16 kD and the expression of mRNA hybridizing with a zein DNA probe. We conclude that an abrupt increase in auxin induces cellular differentiation events in endosperm, including endoredupliction and expression of particular zein storage proteins.     

Low-solubility glycerol dehydratase,a chimeric enzyme of coenzyme B<Subscript>12</Subscript>-dependent glycerol and diol dehydratases

Coenzyme B₁₂-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the β and γ subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the β and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the β and γ subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase β subunit in addition to the diol dehydratase-specific extra-regions of β and γ subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short N-terminal sequences are sufficient to change the solubility of the enzyme.