Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and found to be very similar to that of the nonspecific lipid transfer protein from bovine and rat liver with, as main feature, the absence of arginine, histidine and tyrosine. By way of a specific enzyme immunoassay using affinity-purified antibodies, the levels of nonspecific lipid transfer protein were determined in human livers. Levels varied from approximately 150 ng nonspecific lipid transfer protein per mg 105,000 X g supernatant protein for juvenile and adult humans to 40 ng per mg supernatant protein for a young infant. Levels of nonspecific lipid transfer protein in livers of infants with cerebro-hepato-renal (Zellweger) syndrome were extremely low (i.e., 2 ng per mg supernatant protein). Immunoblotting revealed the presence of crossreactive proteins of molecular masses of 40,000 and 58,000. The 40 kDa and 58 kDa proteins occurred in control livers, whereas only the 40 kDa protein was present in Zellweger livers. As in rat the 58 kDa protein could be demonstrated in a peroxisomal preparation isolated from an adult liver. A possible link between the occurrence of nonspecific lipid transfer protein and the presence of peroxisomes is discussed.     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Characterization of vitellin,egg-specific protein and 30 kDa protein from Bombyx eggs,and their fates during oogenesis and embryogenesis

Three major yolk proteins, vitellin, egg-specific protein and 30 kDa proteins, were purified from the same extracts of Bombyx mori eggs by high-performance liquid chromatography on a molecular sieving column. Each preparation was judged to be homogeneous by polyacrylamide gel electrophoresis. The subunit structure was estimated to be as follows: vitellin is a tetramer with a molecular mass of 420 kDa, consisting of two heavy subunits (178 kDa) and two light subunits (43 kDa); egg-specific protein is a trimer (225 kDa) of two heavy subunits (72 kDa) and one light subunit (64 kDa); 30 kDa proteins are a mixture of three monomers (1, 2 and 3) consisting of respective subunit molecular masses of 32.0, 31.0 and 29.5 kDa. The three yolk proteins contained the usual amino acids together with various lipids and carbohydrates. Antisera to each protein did not cross-react. The titration of vitellin, egg-specific protein and 30 kDa proteins on rocket immunoelectrophoresis showed a differential accumulation pattern during the course of oogenesis. In newly laid eggs, vitellin, egg-specific protein and 30 kDa proteins accounted for approx. 40%, 25% and 35%, respectively, in weight. The eggs developed in male hosts after implantation of ovary discs were deficient in vitellin but contained egg-specific protein and 30 kDa proteins at comparable levels to the normal female eggs. During embryogenesis, egg-specific protein was rapidly and completely utilized. Approx. 35% vitellin and 50% 30 kDa proteins remained unused and were carried over to the hatched larvae. Such accumulation and utilization of yolk proteins are correlated with the fates of the proteins during oogenesis and embryogenesis of B. mori.     

Presence of alpha1-antitrypsin and transferrin in human follicular fluid--correlation with fertilization

We purified two proteins with molecular masses of approximately 50 kDa and 80 kDa with N-terminal sequences similar to those of alpha1-antitrypsin (a1AT) and transferrin indicating that they are identical to or highly homologous to these proteins. Proteins from human follicular fluid were purified after ammonium sulfate fractionation followed by water dialysis and High Performance Liquid Chromatography. The fraction of peak 3 showed a single band on electrophoresis and its N-terminal amino acid sequence was similar to that of human serum transferrin. The fraction of peak 10 proved to be a glycoprotein and its N-terminal amino acid sequence was similar to that of human serum a1AT. There are indications that transferrin may be involved in the fertilization process. Sperm motion was assessed employing computer-assisted semen analysis. The addition of purified protein to prepared sperm samples from normospermic men significantly increases the straight-line velocity (VSL), the amplitude of lateral head displacement (ALH) and the number of progressively motile sperm. a1AT does not seem to have a stimulatory effect on sperm motility.     

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to pI, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.     

Purification of three related peripheral membrane proteins needed for vesicular transport

We report conditions under which Golgi membranes depleted of peripheral membrane proteins can be reconstituted for intra-cisternal vesicular transport. Analysis of the reconstitution reveals requirements for N-ethylmaleimide-sensitive fusion protein, a purified peripheral protein involved in the fusion stage of vesicular transport, as well as other peripheral protein activities which can be provided by mammalian cytosol but not yeast cytosol. The restorative activity in bovine brain cytosol is found in two broad and complementing fractions, of average native molecular masses of about 500 and 40 kDa, termed Fr1 and Fr2, respectively. This resolved transport system was used to develop a purification scheme for Fr2. Three proteins of apparent molecular masses of 35, 36, and 39 kDa (Fr2-alpha, -beta, and -gamma, respectively) were found to be responsible for Fr2 activity and were purified to homogeneity. Each Fr2 protein has activity by itself in the reconstituted in vitro Golgi transport assay, although each exhibits a different specific activity and plateau value. No synergy of the three Fr2 proteins was observed during mixing experiments. The three Fr2 proteins seem to be closely related based on size, in vitro activities, chromatographic properties, and peptide maps and may comprise a new family of proteins involved in vesicular transport.     

Human urine proteome analysis by three separation approaches

The urinary proteome is known to be a valuable field of study related to organ functions. There have been several extensive urine proteome studies. However, the overlapping rate among different studies is relatively low. Whether the low overlapping rate was caused by different sample sources, preparation, separation and identification methods is unknown. Moreover, low molecular mass (<10 kDa) proteins have not been studied extensively. In this report, male and female pooled urine samples were collected from healthy volunteers. The urinary proteins were acetone precipitated, separated and identified by three approaches, 1-DE plus 1-D LC/MS/MS, direct 1-D LC/MS/MS and 2-D LC/MS/MS. 1-D tricine gels were used to separate low molecular mass proteins. The tandem mass spectra of positive identifications were quality controlled both by manual validation and using advanced mass spectrum scanner software. A total of 226 urinary proteins were identified; 171 proteins were identified by proteomics approach for the first time, including 4 male-specific proteins. Twelve low molecular mass proteins were identified. Most urinary proteins had a molecular mass between 30 and 60 kDa and a pI between 4 and 10. The apparent molecular masses of many proteins were different from theoretical ones, which indicated their post-translational modification and degradation. The effects of sample preparation, separation and identification methods on the overlapping rate of different experiments are discussed.     

Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

The flagellar hook–basal body (HBB) complex of the Gram-positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram-negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium . The hook protein had an apparent molecular mass of 29 kDa, and its N-terminal sequence was identical to that of B. subtilis FlgG, which was previously reported as a rod protein. The sequence of the reported FlgG protein of B. subtilis is more closely related to that of FlgE (the hook protein) rather than FlgG (the rod protein) of S. typhimurium , in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa). The hook–basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation. The N-terminus of each of these proteins was sequenced. Comparison with protein databases revealed the following polypeptide–gene correspondences: 82 kDa, fliF ; 59 kDa, flgK ; 35 kDa, orfF ; 32 kDa, yqhF ; 23 kDa, orf3 of the flaA locus; 20 kDa, flgB and flgC ; 13 kDa, not determined. The band at 20 kDa was a mixture of FlgB and FlgC, as revealed by two-dimensional gel analysis. Characteristic features of B. subtilis HBB are discussed in comparison with those of S. typhimiurium .     

Beta-galactosidase of Penicillium chrysogenum: production, purification, and characterization of the enzyme

Intracellular beta-galactosidase from Penicillium chrysogenum NCAIM 00237 was purified by procedures including precipitation with ammonium sulfate, ion-exchange chromatography on DEAE-Sephadex, affinity chromatography, and chromatofocusing. These steps resulted a purification of 66-fold, a yield of about 8%, and a specific activity of 5.84 U mg(-1) protein. Some enzyme characteristics were determined using o-nitrophenyl-beta-d-galactopyranoside as substrate. The pH and temperature optimum of the activity were about 4.0 and 30 degrees C respectively. The K(m) and pI values were 1.81 mM and 4.6. beta-Galactosidase of P. chrysogenum is a multimeric enzyme of about 270 kDa composed of monomers with a molecular mass of 66 kDa.     

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.     

蛋白激酶组在玉米叶片ABA和H2O2诱导抗氧化防护中的作用

蛋白磷酸化在植物细胞脱落酸（ABA）介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和Ca2+依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa, 52kDa, 49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用, 而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize

Protein phosphorylation plays a central role in mediating abscisic acid (ABA) signaling transduction in plant cells, whereas many of the sensory proteins involving in ABA signaling pathway remain unclear. Here, using a modified in vitro kinase assay, our results showed that ABA and H2O2 induced a rapid activation of total protein kinases and calcium dependent protein kinases in the leaves of maize seedlings. However, ABA-induced activation of protein kinases was inhibited by reactive oxygen species (ROS) inhibitors or scavengers. Protein kinase inhibitors decelerated not only the ABA and H2O2 -induced kinase activity but also ABA or H2O2-induced antioxidant enzyme activity. Protein phosphorylation caused by ABA and H2O2 preceded ABA or H2O2 -induced antioxidant defense obviously. Using in-gel kinase assays, our results showed that several protein kinases with molecular masses of 66kDa, 52kDa, 49kDa and 35kDa respectively might mediate ABA and H2O2-induced antioxidant defense. And the 66kDa and 49kDa protein kinases may act downstream of ROS, and the 52kDa and 35kDa protein kinases may act between ABA and ROS in ABA-induced antioxidant defensive signaling.     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes.

1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had an apparent molecular mass of 18 kDa. Results of heparitinase digestion and HNO2-treatment indicated a clustering of sulfate groups in the distal part of the HS side chains. 5. Peptide mapping after trypsin, clostripain or V8 protease digestion of radiolabeled human and equine heparan sulfate proteoglycans (HSPG) preparations with three different separation techniques showed large differences. 6. Polyclonal antisera raised against the HSPGs reacted against the core proteins. Both HSPG preparations and their antisera showed ca 40% cross-reactivity. About 50% of monoclonal antisera elicited against one HSPG preparation showed reaction with both HSPG preparations. 7. Polyclonal antisera stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies of kidney sections from horse, man and various mammalian species. 8. Biochemical and immunological data indicate that HSPGs from equine and human GBM have a comparable structure, but the core proteins differ considerably.     

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

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

Biochemical characterization and N-terminal sequences of two new trypsin inhibitors from Copaifera langsdorffii seeds

Two new trypsin inhibitors, TDI-I and TDI-II, were purified from the seeds of the native Brazilian tree Copaifera langsdorffii (Caesalpinoideae, Leguminosae). The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on trypsin-Sepharose, and reversed-phase (RP) HPLC. RP-HPLC yielded two forms (TDI-I and TDI-II), as confirmed by isoelectric focusing, with pI values between 7.0 and 8.1. The molecular mass of the TDI forms was 24 kDa based on FPLC gel filtration on Superdex 75. Under reducing conditions in tricine SDS-PAGE the molecular masses of TDI-I and TDI-II were 12 and 10 kDa, respectively. The Ki values were 1.1 and 1.2 nM for TDI-I and TDI-II, respectively, and there was no inhibitory effect on chymotrypsin. Amino acid analysis revealed high levels of aspartic acid, glutamic acid, serine, glycine, proline, and lysine but low levels of methionine and aromatic amino acids in both inhibitors; the calculated molecular masses were 11,456 and 10,008 for TDI-I and II, respectively. Based on the N-terminal sequences of TDI-I and TDI-II, TDI-I belongs to the Kunitz family of trypsin inhibitors, whereas TDI-II showed no homology to any other protein. This observation suggests that TDI-II belongs to a new inhibitor subclass of low-molecular mass proteins in the subfamily Caesalpinoideae.     

蛋白激酶组在玉米叶片ABA和H202诱导抗氧化防护中的作用

蛋白磷酸化在植物细胞脱落酸（ABA）介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和ca^2＋依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa,52kDa,49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用,而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

Purification and characterisation of gelonin from seeds of Gelonium multiflorum

Gelonin, a ribosome-inactivating protein has been isolated from the seeds of Gelonium multifluorum of Euphorbiaceae family by two methods and the results are compared. In method-I conventional aqueous extraction, cation-exchange and gel-filtration chromatography has been used. In method-II S-Sepharose fast flow gel has been used to purify the proteins from the seed extract, followed by ammonium sulfate fractionation, cation-exchange and gel-filtration chromatography. Extensive physico-chemical and immunological characterizations show that molecular weight of gelonin as determined by gel-filtration chromatography and SDS-PAGE is approximately 30 kDa. The non-proteinous material which binds to CMC-gel in association with gelonin in method-I is substantially removed when gelonin is purified by method-II. Cation exchange, G-100 chromatography, RP-HPLC and SDS-PAGE show that method-II yields 50% more purified gelonin when compared to the yield by method-I. The immunoreactivity of gelonin obtained by methods I and II vary from 22-26% and 50-66% respectively and the ribosome-inactivating property vary from 46-56% and 70-87% respectively.