Major Polypeptides Research Articles

Spermidine-binding proteins. Purification and expression analysis in maize.

Polyamine-binding proteins have been identified in a wide range of organisms, including mammals, yeasts, and bacteria. In this work, we have investigated specific spermidine binding to plant membrane proteins purified from microsomes of etiolated maize (Zea mays) coleoptiles. In the final purification step, specific spermidine-binding activity (K(d) 6.02 10(-7) M) was eluted from a HiTrapQ fast-protein liquid chromatography column at about 0.25 M NaCl, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most active fraction showed a major polypeptide of about 60 kD and another copurifying 18-kD protein. Competition experiments, performed on HiTrapQ active fractions, confirmed the specificity of the binding. Upon Sephadex G-100 gel filtration, spermidine binding was associated almost exclusively with the 18-kD protein. On the basis of the N-terminal sequences, degenerate oligonucleotide probes were designed and used to isolate, by reverse transcriptase-polymerase chain reaction and polymerase chain reaction, cDNA fragments of about 1 kb for the 60-kD protein, and 0.9 kb for the 18-kD protein. Northern-blot analysis performed on etiolated coleoptiles and different tissues from 10-d-old maize plants indicated the presence of two different mRNAs of 1.7 and 0.7 kb. Southern-blot analysis indicated that the genes encoding the 60- and 18-kD proteins are probably derived from differential processing of the same precursor mRNA. Using rabbit polyclonal antibodies raised against these proteins, affinity purification and dot-blot experiments detected analogous membrane proteins in monocot and dicot plants.

Deregulated expression of the PCPH proto-oncogene in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene.

The PCPH proto-oncogene was identified by its frequent activation in Syrian hamster fetal cells exposed to 3-methylcholanthrene. We previously isolated human PCPH cDNA and studied its expression in normal human tissues. We report herein the pattern of PCPH expression in normal rat tissues. Each tissue expressed one major PCPH polypeptide that varied in molecular mass in different tissues. Normal mammary gland expressed a single PCPH polypeptide of 27 kDa. This PCPH form also was expressed in lactating mammary glands but at significantly greater levels. These results suggest the existence of tissue-specific regulatory mechanisms for PCPH expression that may be influenced by the differentiation stage. Our previous studies on the involvement of PCPH in human cancer showed that human breast tumor cell lines have frequent alterations in PCPH, including multiple PCPH polypeptide forms that are not expressed in normal cells. These cell lines also have frequent loss of a 27-kDa form identified as the only PCPH polypeptide expressed by normal human breast epithelial cells. In this study, we found that these same alterations occurred in vivo during mammary carcinogenesis in Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene, in both benign and malignant tumors, indicating that stable changes in PCPH expression took place early in the neoplastic process. Results showed that this experimental system is relevant to human breast carcinogenesis and provides an excellent model to study the molecular basis of the regulation of PCPH expression during normal differentiation and pathologic stages of neoplasia of the mammary gland and to analyze the role of PCPH in the carcinogenic process. Furthermore, the detection of atypical PCPH polypeptides in tumors suggests that PCPH immunodetection may be applied as a diagnostic tool for the early identification of neoplastic breast epithelial cells.

Metal ions restore the proteolytic resistance of denatured conglutin gamma, a lupin seed glycoprotein, by promoting its refolding.

The susceptibility to trypsin of conglutin gamma, a lupin seed glycoprotein affected by this enzyme only when in a non-native conformation, was used to study the effect of Zn(2+) and other metal ions on the structural dynamics of the protein. When acid-treated trypsin-susceptible conglutin gamma was incubated at neutral pH in the presence of Zn(2+), it became resistant to tryptic attack, contrary to the protein treated in the absence of Zn(2+). The time course of this refolding event has been quantitatively evaluated by SDS-PAGE. Amino acid sequencing of the major polypeptide fragments, produced by trypsin before completion of the refolding process, indicated that only a few cleavable bonds were accessible to the enzyme. This suggested that the presence of metal ions affected the pathway of degradation of the protein, by inducing its folding. Among the other metal ions tested, Ni(2+) also promoted the adoption of a trypsin-resistant conformation of conglutin gamma, whereas Mn(2+) and Ca(2+) had only much lower effects. The relevance of these findings for a deeper understanding of the in vivo degradation of plant food proteins and how it is affected by metal ions are discussed.

Characterisation of a pathogenic virus isolated from marine threadfin fish ( Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin ( Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70–80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 nm in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 °C). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 °C), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass ( Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass.

The 23-kDa polypeptide from Common Frog lens: isolation, characteristics and cellular localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23-kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight of more than 300 kDa (alpha-crystallin fraction). A highly pure fraction of the 23-kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23-kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighbouring the plasma membranes in cortical fibers. The 23-kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23-kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23-kDa polypeptide and the relation of its expression with lens cell differentiation are discussed.

In Vivo Properties of Phosphoenolpyruvate Carboxylase in Crassulacean Acid Metabolism Plants. Is Pineapple CAM Not Regulated by PEPC Phosphorylation?

The in vivo sensitivity to inhibition by L-malate of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in rapidly prepared crude extracts (within 2 min) was investigated for two CAM plants Kalanchoe pinnata and pineapple (Ananas comosus (L.) Merr. cv. Smooth-cayenne N67-10) . The phosphorylation process may not occur in pineapple PEPC. Variations were also observed in apparent kinetic properties and molecular mass as well as subunit composition of PEPC from these plants. Two different subunits (major polypeptide 112 kDa and minor polypeptide 119 kDa) were separated from day and night forms of PEPC from K. pinnata but only one identical subunit (107 kDa) from pineapple. The integrity of both forms of in vivo PEPC from these plants was demonstrated by Western blot analyses using anti-N-terminus antibody. K. pinnata lost malate sensitivity about 87% whereas pineapple PEPC lost only 21% sensitivity at night in the presence of 2 mM L-malate. The apparent Ki (L-malate) was 6.4-fold higher in phase I (5.8 mM) than that in phase III (0.9 mM) from K. pinnata but it was only 1.0 and 0.5 mM for day and night PEPC respectively from pineapple. These findings suggest phosphorylation process may not be the major regulatory mechanism in pineapple PEPC.

Assessing the relative importance of light and the circadian clock in controlling chloroplast translation in Chlamydomonas reinhardtii.

Previous work has shown that transcription of a number of chloroplast-encoded genes, including those for photosynthesis, are under circadian clock control in Chlamydomonas reinhardtii. However, some of these genes encode long-lived mRNAs that are also subject to translational control. Rates of synthesis of the major chloroplast translation products vary dramatically (10-20-fold) during light-dark (LD) cycles, peaking in the light period. To determine whether this pattern reflects circadian clock control, LD-grown cells were shifted to continuous light (LL) and chloroplast protein synthesis monitored by periodic pulse-labeling in the presence of cycloheximide; chloroplast protein synthesis in LD was also examined for comparison. The LD patterns of synthesis of the major polypeptides (including D1, D2, and the large subunit of ribulose-1,5-bisphosphate carboxylase (LS)) were similar to those obtained previously in the absence of cycloheximide. In the LL condition, rates of synthesis of the major chloroplast translation products were high throughout the period examined ( approximately 36 h), fluctuating > 3-fold, although they were generally higher in the subjective light period. LD-grown cells were also shifted to continuous dark (DD) and chloroplast protein synthesis analyzed for approximately 24 h starting from the mid-dark period. There was a gradual decline in synthesis of the major proteins during the first subjective light period, which was followed by a very small peak in synthesis around the second subjective dark --> light transition. RNA blot analysis showed that the mRNAs for D1, D2 and LS were present at high levels during the period of declining translation. These results indicate that with photoautotrophic growth in LD cycles, the illumination conditions per se are more important than the clock in determining chloroplast translation, but the clock may contribute to this regulation. The advantages of controlling translation by a direct light response and transcription primarily by the circadian clock are discussed. Finally, evidence of translational control of elongation factor Tu synthesis was obtained.

Human cell DNA replication is mediated by a discrete multiprotein complex.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.

Characterization of an Extracellular Serine Protease of Fusarium eumartii and its Action on Pathogenesis Related Proteins

Proteases have been proposed as part of the invasion strategies of some pathogenic fungi. In this work, a serine protease produced by the phytopathogenic fungus Fusarium solani f. sp. eumartii was purified and characterized. Purification of the enzyme was accomplished by gel filtration through a Superose 12 column, followed by hydrophobic interaction chromatography in Phenyl Superose and gel filtration chromatography through Superdex 75. Analysis of the purified enzyme by SDS/PAGE without heat treatment, revealed a single band, which corresponded to the proteolytic activity detected by zymogram. When this protein was subjected to denaturing conditions, two major polypeptides of approximately 30 and 33 kDa were revealed. The N-terminal amino acid sequence of one of these polypeptides showed a high similarity with fungal mature serine proteases of the subtilisin family. This protease hydrolysed in vitro, specific polypeptides of potato intercellular washing fluids and cell walls. The protease was also able to degrade pathogenesis-related proteins from the intercellular washing fluids. The role of this serine protease as part of the fungal strategy to colonize potato tuber tissues is discussed.

Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae).

Two lytic viruses specific for Chrysochromulina ericina (Prymnesiophyceae) and for Pyramimonas orientalis (Prasinophyceae) were isolated from Norwegian coastal waters in June 1998. The lytic cycle was 14-19 h for both viruses; the burst size was estimated at 1800-4100 viruses per host cell for the Chrysochromulina virus and 800-1000 for the Pyramimonas virus. Thin sections of infected cells show that both viruses replicate in the cytoplasm and that they have a hexagonal cross section, indicating icosahedral symmetry. The Chrysochromulina virus had a particle size of 160 nm and a genome size of 510 kbp; the size of the major polypeptide was 73 kDa. The Pyramimonas virus had a particle size of 220 x 180 nm and a genome size of 560 kbp; the size of the major polypeptide was 44 kDa. The genome sizes of these viruses are among the largest ever reported for viruses and they are larger than the minimum required for cellular life. The Chrysochromulina virus clone CeV-01B and the Pyramimonas virus clone PoV-01B described in this study have several properties in common with other viruses infecting microalgae, suggesting that they belong to the Phycodnaviridae.

Protein classification of beach pea (Lathyrus maritimus L.)

Abstract Protein fractionation of whole seed, cotyledons and hulls of beach pea (Lathyrus maritimus L.) was carried out. Surface topography of pea protein isolates and protein fractions, as well as their polyacrylamide gel electrophoresis (PAGE), was studied. The nitrogen solubility of beach pea seed meal was minimum at pH 4.5. Globulin was the major protein fraction present and its content in whole seeds (57%), cotyledons (62%) and hulls (24%) of beach pea was lower than those of other common pea cultivars; the same was true for its albumin content. However, glutelin content in beach pea seed and its parts (cotyledons and hulls) was higher than those of other pea cultivars. The albumin fraction contained the highest amount of total sulphur-containing amino acids followed by glutelin, globulin and prolamine. The amount of sulphur-containing amino acids in beach pea protein fractions was higher than in other peas. Predicted biological value of albumin and glutelin fractions of beach pea was also higher than green pea and grass pea. Beach pea seed protein fractions showed different UV spectra from other pea cultivars. Protein isolate and protein fractions of beach pea seeds showed similar topographical characteristics to those of other peas. Major polypeptide bands in the range of 35–47 kDa in the protein isolate, as well as protein fractions for beach pea, were detected by PAGE.

Characterization and immunological analysis of ferritin from the hemolymph of Galleria mellonella

Ferritin, an iron-binding protein, was purified from the larval hemolymph of the wax moth, Galleria mellonella by KBr density ultracentrifugation and FPLC (Superose 6). The iron content of ferritin was determined by atomic emission spectroscopy and Ferene S stain. Native molecular mass of ferritin was estimated as 630 kDa. SDS-PAGE revealed that the ferritin consists of two major polypeptides of 26 and 32 kDa and one minor polypeptide of 30 kDa. An isoelectric point of ferritin was measured to be approximately 7.3 and only the 32-kDa subunit is glycosylated. The ferritin contains large amounts of lysine, glutamine, glutamic acid and leucine but tryptophan was not detected. Electron microscopic examination of negatively stained preparations showed an 11-nm particle in external diameter and 7-nm iron core. Ferritin is present in both the ovary and testis. Localization of ferritin by immunoelectron microscopy in ovary and testis revealed that the gold particles were located in vitelline membrane and yolk granules but not in follicular epithelium of ovary. In the testis, the gold particles were located in testicular fluid and lumen of vas deferens.

Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9

Ly-9 is a mouse cell-surface glycoprotein that is selectively expressed on thymocytes and on mature T and B lymphocytes. Ly-9 belongs to the CD2 subset of the immunoglobulin superfamily, an emerging family of cell signaling receptors. Recently, a partial human Ly-9 complementary DNA (cDNA) sequence has been described. Full-length cDNA clones were isolated that included the initiation codon, the sequence encoding the full signal peptide, and 14 amino acids more in the cytoplasmic domain than in the previously reported clone. The predicted extracellular domain of human Ly-9 contains 4 immunoglobulinlike domains, similar to those in mouse Ly-9. Northern blot analysis revealed that the human Ly-9 messenger RNA (2.6 kb) is expressed predominantly in lymph node, spleen, thymus, and peripheral blood leukocytes. Four monoclonal antibodies (mAbs) were raised against human Ly-9 by immunizing mice with the pre-B-cell line 300.19 stably transfected with human Ly-9 full-length cDNA. These mAbs strongly stained the surfaces of cells transfected with human Ly-9 cDNA but not of untransfected cells. Human Ly-9 expression was restricted to T and B lymphocytes and thymocytes, with the highest levels of expression on CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. Monocytes, granulocytes, platelets, and red blood cells were uniformly negative for Ly-9. These mAbs immunoprecipitated major polypeptides of 120 kd from the transfected cells and 120 kd and 100 kd from B-cell line Daudi, probably because of the cell-surface-expressed isoforms. These data demonstrate that human Ly-9 is a new marker for the study of normal and malignant leukocytes. (Blood. 2001;97:3513-3520)

Characterization of proteins in the outer membrane preparation of a murine pathogen, Helicobacter bilis.

Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins from H. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.

Identification of a Truncated Form of the G-protein Regulator AGS3 in Heart That Lacks the Tetratricopeptide Repeat Domains

AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.

Biophysical Studies on the RNA Cores of Satellite Tobacco Mosaic Virus

Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90°C. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2–12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.

Influence of benomyl on photosynthetic capacity in soybean leaves

This investigation was performed to study the influence of benomyl on photosyn- thetic pigments and enzymes in soybean leaves. Chlorophyll and pheophytin levels were re- duced by benomyl 45 days after greening. These results indicate that chlorophyll a and b, and pheophytin must be controlled by benomyl. SDS-PAGE analysis showed that 50 and 14.5 kD polypeptides represented as the large and small subunits of rubisco. In the both of these sub- units, the band intensity of the control was significantly higher than that after benomyl treatment, indicating that these two subunits are affected by benomyl. Benomyl strongly in- hibited both the activity and content of rubisco as its concentration was gradually increased. However, it remains unclear whether this reduction of rubisco level was due to a reduced level of rubisco activase. Two major polypeptides of 46 and 42 kD were identified as rubisco activase subunits by SDS-PAGE. The intensity of these two bands was shown to be higher in the control than after benomyl treatment. These results indicate that the rubisco decrease resulting from increased benomyl concentrations was caused by rubisco activase. A significant decrease in both the activity and content of rubisco activase by benomyl was also observed. These results suggest that the decrease in rubisco level caused by benomyl is accompanied by a decrease in both the activity and content of rubisco activase.

MOLECULAR CHARACTERIZATION OF THE ?-CONGLUTIN OF LUPIN SEEDS

β-conglutin from Lupinus campestris was extracted and purified by gel filtration chromatography. The protein with molecular mass around 190 kDa showed by SDS-PAGE three major polypeptides of 53, 60 and 63 kDa and a minor polypeptide of 30 kDa. Isoelectric focusing of the native protein gave three isoforms with isoelectric points of 6.2, 6.3 and 6.4. Its sedimentation coefficient was 8.0S, with a hydrodynamic diameter of 110 Å and possessed antigenic determinants in common with 7S proteins from common bean and soybean.

A HECT Domain E3 Enzyme Assembles Novel Polyubiquitin Chains

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.

Polypeptide profile and functional properties of defatted meals and protein isolates of canola seeds

Laboratory-defatted meals from four types of canola seeds were analysed for protein profile by reducing and non-reducing forms of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In the absence of a reducing agent (2-mercaptoethanol), four major polypeptide bands (16, 18, 30 and 53 kDa) were prominent in similar ratios in all varieties. In the presence of mercaptoethanol, significant reductions in intensity of the major bands occurred, suggesting that the major polypeptides contained smaller units which were held together by disulphide bonds. Meals from Brassica napus seeds had higher protein solubilities than meals from Brassica rapa seeds. Meals with higher protein solubility values also had higher foaming capacity (FC) values. Generally, the acid-precipitated (pH 4.0) protein isolates (APPIs) had higher FC values than the calcium-precipitated isolates (CPPIs). On the other hand, the CPPIs formed emulsions with higher values of emulsifying activity index (EAI) when compared to the APPIs. The results indicate that variations in functional properties of protein isolates and meals between the four seed types were probably due to differences in protein conformation in aqueous solutions rather than differences in polypeptide composition. © 2001 Society of Chemical Industry