Comigration of two autophagosome-associated dehydrogenases on two-dimensional polyacrylamide gels

Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles.     

A monoclonal antibody that inhibits translation in Sf21 cell lysates is specific for glyceraldehyde-3-phosphate dehydrogenase

Monoclonal antibody (Mab) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to be specific for a 60-kDa form of elongation factor-1 alpha (EF-1alpha), primarily because the antigen immunoprecipitated by Mab 8B7 cross-reacted with Mab CBP-KK1, an antibody generated to EF-1alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded an additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mab 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pI 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mab 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation.     

Mammalian FKBP-25 and its associated proteins

Soluble proteins from porcine brain were divided into two packs: (1) proteins which pass freely through CM52-cellulose, and (2) proteins retained on CM52. Each of these two packs of proteins was fractionated on preparative flat-bed isoelectrofocusing gel in the range of pH 2-12. Native FKBP-25 and its truncated forms were found among other proteins retained on CM52-cellulose. Immunoblotting with anti-FKBP-25 showed two bands in the range 27-30 kDa, one due to unmodified FKBP-25 and other due to FKBP-25 mixed with high-mobility group II protein (HMG-II). Selective immunostaining with anti-FKBP-25 antibodies of proteins which were not retained on CM52-cellulose showed several bands within the range of pI 7-5 and mass of 23 +/- 2 kDa. These fractions of proteins were next resolved on two-dimensional gels and immunostained with anti-FKBP-25 antibodies. Six proteins in the pI range 7-5 were detected. Edman degradation of alpha-chymotrypsin digests of the major spot suggests that it contains the GTP-binding protein Rab5 co-migrating with guanylyl kinase, whereas MALDI-TOF showed that a residual content of FKBP-25 may be also associated with these two proteins. A residual quantity of FKBP-25 was also associated with the phosphatidylethanolamine-binding protein which is abundant in the brain.     

Molecular heterogeneity of pro-atrial natriuretic factor

Analysis by two-dimensional gel electrophoresis and Western blotting of the atrial natriuretic factor (ANF) content of atrial granules revealed the presence of at least 15 immunoreactive spots whose molecular mass distribution ranged from 16.8 to 35 kDa and their pI values from 5.12 to 5.98. About 90% of the immunoreactive ANF material was contained within four spots (spot 1: 34.8 kDa, pI 5.67; spot 5: 16.8 kDa, pI 5.50; spot 6: 16.8 kDa, pI 5.67; spot 7: 16.8 kDa, pI 5.98). Investigation of the molecular nature of spot 1 indicated that it is a dimer of pro-ANF since it possesses the same immunoreactivity, the same charge, double its mass, and can be converted with dithiothreitol into a 16.8-kDa pro-ANF form. Alkaline phosphatase and protein kinase A treatments indicated that spots 5, 6, and 7 are probably not phosphorylated forms of pro-ANF. Carboxypeptide A and B treatments in conjunction with amino acid analysis suggested that spot 7 is ANF-(1-128); spot 6, the major one, ANF-(1-126); and spot 5, ANF-(1-123) or ANF-(1-124). Water deprivation or morphine injection, two maneuvers which are known to influence ANF secretion and atrial ANF content, failed to affect the molecular heterogeneity of pro-ANF except for spot 1. The formation of the dimer appeared to be time-dependent. These results emphasize the heterogeneity of the pro-ANF molecule stored in atrial granules. We suggest that this heterogeneity may be due, in part, to the action of some proteases, such as carboxypeptidase E or a tripeptidyl carboxyhydrolase.     

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from European pilchard Sardina pilchardus

The NAD＋-dependent cytosolic glyceralehyde-3-phosphate dehydrogenase （GAPDH; EC 1.2.1.12） was purified from the skeletal muscle of European pilchard Sardina pilchardus and its physicochemical and kinetic properties were investigated. The purification method consisted of two steps, ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography, resulting in an approximately 78-fold increase in specific activity and a final yield of approximately 25%. The Michaelis constants （Kin） for NAD＋ and D-glyceraldehyde-3-phosphate were 92.0 μM and 73.4 μM, respectively. The maximal velocity （Vmax） of the purified enzyme was estimated to be 37.6 U/mg. Under the assay conditions, the optimum pH and temperature were 8.0 and 30 ℃. The molecular weight of the purified enzyme was 37 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yielding a molecular weight of 154 kDa suggested that the enzyme is a homotetramer. Polyclonal antibodies against the purified enzyme were used to recognize the enzyme in different sardine tissues by Western blot analysis. The isoelectric point, obtained by an isoelectric focusing system in polyacrylamide slab gels, revealed only one GAPDH isoform （pI 7.9）.     

Purification and identification of an IgE suppressor from strawberry in an in vitro immunization system

We purified and identified an IgE suppressor from the strawberry 'Toyonoka', based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15-48 kDa range and <10 kDa have an IgE suppressive activity. Furthermore, the fraction in 15-48 kDa was subjected to chromatofocusing and found to have activities at isoelectric points, pI 6.0, 7.0, and 8.0-9.2. We focused on the active fractions of pI 8.0-9.2 and the purified a large amount of strawberry extracts by cation exchange resins in batch. A purified 39 kDa protein showed homology to plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in N-terminal amino acid sequence and had GAPDH enzymatic activity. Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone of the protein matched with the sequence of Fragaria x ananassa GAPDH in the GenBank with >98% identical nucleotides and >99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.     

Characterization of purified hepatitis B surface antigen containing pre-S(2) epitopes expressed in Saccharomyces cerevisiae

The cloning and expression of the hepatitis B middle-protein surface antigen gene in the yeast Saccharomyces cerevisiae is described. A generalized expression vector carrying the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter was used. Expressed material, in the form of supramolecular particles, was purified and characterized. Severe proteolysis within the pre-S(2) region was observed for material expressed in a wild-type yeast host. This proteolysis was substantially reduced by utilization of a protease-deficient host. Immunoblotting of sodium dodecyl sulfate-polyacrylamide gels with several antibodies of differing specificity was performed to characterize the various protein species present. All species were analyzed by N-terminal sequencing after electroelution from gels. Carbohydrate staining of gels and glycosidase treatments of the purified antigen material indicated that full-length antigen was present in both glycosylated and unglycosylated forms. Glycosylation appeared to be of both asparagine-linked and threonine/serine-linked types. Site-directed mutagenesis was used to convert two arginine residues in the pre-S(2) region of the antigen to glutamine residues. The changes abolished reactivity with one polyclonal and two monoclonal antibodies specific for epitopes within the pre-S(2) region.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Submicromolar concentrations of palmitoyl-CoA specifically thioesterify cysteine 244 in glyceraldehyde-3-phosphate dehydrogenase inhibiting enzyme activity: a novel mechanism potentially underlying fatty acid induced insulin resistance

The accumulation of fatty acids and their metabolites results in insulin resistance and reduced glucose utilization through a variety of complex mechanisms that remain incompletely understood. Herein, we demonstrate that submicromolar concentrations of palmitoyl-CoA inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) enzyme activity through the covalent thioesterification of palmitate to GAPDH. First, incubation of GAPDH with palmitoyl-CoA (0.5-5 microM) resulted in the dramatic concentration-dependent inhibition of GAPDH enzyme activity. Second, incubation of GAPDH with [(14)C]palmitoyl-CoA followed by SDS-PAGE and autoradiography identified a covalently radiolabeled adduct present at approximately 35 kDa with a stoichiometry of one molecule of palmitoyl-CoA per GAPDH tetramer. Third, mass spectrometric analyses of intact GAPDH treated with palmitoyl-CoA demonstrated the covalent addition of palmitate to the GAPDH protein. Fourth, trypsinolysis of the modified protein revealed that the peptide (232)VPTPNVSVVDLTRC*R(245) was covalently modified. Fifth, the site of palmitoylation was demonstrated to be Cys-244 by analyses of product ion mass spectra. These assignments were further substantiated using different molecular species of acyl-CoAs resulting in the anticipated changes in both the masses of adduct ions and their fragmentation patterns. Sixth, GAPDH palmitoylation was demonstrated to facilitate the translocation of GAPDH to either lipid vesicles or naturally occurring biologic membranes. Since the hallmark of lipotoxicity is the accumulation of fatty acids and their acyl-CoA metabolites in excess of a cell's ability to appropriately metabolize them, these results identify a novel mechanism potentially contributing to the insulin resistance, reduced glucose utilization, and maladaptive metabolic alterations underlying the lipotoxic state.     

Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells.

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.     

Functional studies of chloroplast glyceraldehyde-3-phosphate dehydrogenase subunits A and B expressed in Escherichia coli: formation of highly active A4 and B4 homotetramers and evidence that aggregation of the B4 complex is mediated by the B subunit carboxy terminus

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (phosphorylating, E.C. 1.2.1.13) (GAPDH) of higher plants exists as an A₂B₂ heterotetramer that catalyses the reductive step of the Calvin cycle. In dark chloroplasts the enzyme exhibits a molecular mass of 600 kDa, whereas in illuminated chloroplasts the molecular mass is altered in favor of the more active 150 kDa form. We have expressed in Escherichia coli proteins corresponding to the mature A and B subunits of spinach chloroplast GAPDH (GapA and GapB, respectively) in addition to a derivative of the B subunit lacking the GapB-specific C-terminal extension (CTE). One mg of each of the three proteins so expressed was purified to electrophoretic homogeneity with conventional methods. Spinach GapA purified from E. coli is shown to be a highly active homotetramer (50–70 U/mg) which does not associate under aggregating conditions in vitro to high-molecular-mass (HMM) forms of ca. 600 kDa. Since B₄ forms of the enzyme have not been described from any source, we were surprised to find that spinach GapB purified from E. coli was active (15–35 U/mg). Spinach GapB lacking the CTE purified from E. coli is more highly active (130 U/mg) than GapB with the CTE. Under aggregating conditions, GapB lacking the CTE is a tetramer that does not associate to HMM forms whereas GapB with the CTE occurs exclusively as an aggregated HMM form. The data indicate that intertetramer association of chloroplast GAPDH in vitro occurs through GapB-mediated protein-protein interaction.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - CTE carboxy-terminal extension - HMM high molecular mass - ATP adenosine triphosphate - 3PGA 3-phosphoglycerate - 1,3bisPGA 1,3-bisphosphoglycerate - HMM high-molecular mass     

BSP A1/A2-like proteins in ram seminal plasma

The objective of this study was to assess the protein profile of ovine seminal plasma using 2D-PAGE and verify if BSP A1/A2 are present in ovine seminal plasma. Seminal plasma was collected from three mature rams and pooled to eliminate individual differences. Seminal plasma samples were submitted to 2D-PAGE using 12% acrylamide gels. The image analysis software identified 21 protein spots on the air-dried gel, with molecular weight ranging from 15 to 115 kDa and pI 3.2 to 8.7. The most prominent spots were those <30 kDa. The most intensely stained spots were: 3 (18-19 kDa, pI 4.8-5.0), 5 (17-18 kDa, pI 5.0-5.2), 7 (15-16 kDa, pI 6.2-6.4), and 23 (105-108 kDa, pI 6.8-7.0). Three of these spots (spots 3, 5 and 7, respectively) accounted for 41.1% of the relative intensity of the spots of the gels, based on the intensity of the Comassie blue staining. Western blot analysis indicated that spots 3 and 5 were similar to BSP A1/A2 (16.5, pI 4.7-5.0 and 16 kDa, pI 4.9-5.2) identified in Manjunath's studies [Manjunath P, Sairam MR. Purification and biochimical characterization of three major acid proteins (BSP A1, BSP A2 and BSP A3) from bovine seminal plasma. Biochem J 7 (1987) 685-92.], based on the specific reaction of the polyclonal antibody to those spots.     

Four viral proteins of white spot syndrome virus (WSSV) that attach to shrimp cell membranes

White spot syndrome virus (WSSV) is a major shrimp pathogen that has a widespread negative affect on shrimp production in Asia and the Americas. It is known that WSSV infects shrimp cells through viral attachment proteins (VAP) that bind with shrimp cell receptors. However, the identity of both WSSV VAP and shrimp cell receptors remains unclear. We used digoxigenin (DIG)-labeled shrimp hemocyte and gill cell membranes to bind to WSSV proteins immobilized on nitrocellulose membranes, and 4 putative WSSV VAP (37 kDa, 39 kDa and 2 above 97 kDa) were identified. Mass spectrometric analysis identified the 37 kDa putative VAP as the product of WSSV gene VP281.     

cDNA,genomic sequence cloning and overexpression of glyceraldehyde-3-phosphate dehydrogenase gene (<Emphasis Type="Italic">GAPDH</Emphasis>) from the Giant Panda

GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is a key enzyme of the glycolytic pathway and it is related to the occurrence of some diseases. The cDNA and the genomic sequence of GAPDH were cloned successfully from the Giant Panda (Ailuropoda melanoleuca) using the RT-PCR technology and Touchdown-PCR, respectively. Both sequences were analyzed preliminarily. The cDNA of GAPDH cloned from the Giant Panda is 1191 bp in size, contains an open reading frame of 1002 bp encoding 333 amino acids. The genomic sequence is 3941 bp in length and was found to possess 10 exons and 9 introns. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence are highly conserved in some mammalian species, including Homo sapiens, Mus musculus, Rattus norvegicus, Canis lupus familiaris and Bos taurus. The homologies for the nucleotide sequences of the Giant Panda GAPDH to that of these species are 90.67, 90.92, 90.62, 95.01 and 92.32% respectively, while the homologies for the amino acid sequences are 94.93, 95.5, 95.8, 98.8 and 97.0%. Primary structure analysis revealed that the molecular weight of the putative GAPDH protein is 35.7899 kDa with a theoretical pI of 8.21. Topology prediction showed that there is one Glyceraldehyde 3-phosphate dehydrogenase active site, two N-glycosylation sites, four Casein kinase II phosphorylation sites, seven Protein kinase C phosphorylation sites and eight N-myristoylation sites in the GAPDH protein of the Giant Panda. The GAPDH gene was overexpressed in E. coli BL21. The results indicated that the fusion of GAPDH with the N-terminally His-tagged form gave rise to the accumulation of an expected 43 kDa polypeptide. The SDS-PAGE analysis also showed that the recombinant GAPDH was soluble and thus could be used for further functional studies.     

Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.     

Isolation of a 43 kDa protein from Mycobacterium tuberculosis H37Rv and its identification as a pyridine nucleotide transhydrogenase

R.G. DESHPANDE, M.B. KHAN, D.A. BHAT AND R.G. NAVALKAR. 1994. A 43 kDa protein (TB43) was isolated from the cell sonicate (CS) of Mycobacterium tuberculosis H₃₇Rv with immobilized metal affinity chromatography (IMAC) on a Ni-nitrilotriacetic acid column. Two-dimensional electrophoresis of the IMAC fraction showed a major spot with an M_r of 43000 and a pI of ˜6.0. The N-terminal amino acid sequence of TB43 was met-arg-val-gly-ile-pro-asn-glu-thr-lys-asn-asn-glu-phe-arg-val-ala-ile-thr-pro-ala. It showed 86% homology with the N-terminal end of the alanine dehydrogenase of Myco. tuberculosis and 65% homology with the N-terminal end of the alpha-subunit of the Escherichia coli pyridine nucleotide transhydrogenase (Tsh). TB43 did not show any alanine dehydrogenase activity and did not react with monoclonal antibody (MAb) HBT10, which is known to recognize the 40 kDa alanine dehydrogenase of Myco. tuberculosis. It was also not recognized by MAb F29–29 which is known to react with a 43 kDa protein of Myco. tuberculosis complex. This protein exhibited strong Tsh activity. A similar 43 kDa protein showing Tsh activity was also isolated by IMAC from Myco. bovis CS. However, the pI of the protein was ˜7.0. A similar protein could not be isolated from the CS or culture filtrate of Myco. bovis BCG and Myco. tuberculosis H₃₇Ra. TB43 is a cell-associated pyridine nucleotide transhydrogenase and is distinct from the 40/44 kDa secreted alanine dehydrogenase of Myco. tuberculosis.     

Immunological studies on the respiratory burst oxidase of pig blood neutrophils

Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O2-)-generated oxidase activity, was separated by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316-12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF-PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O2- -generating activity of the membrane-solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (PI 5.0) is one of the component(s) of the O2- -generating system. The enzyme gave rise to a band corresponding to a major protein of 72 +/- 4 kDa on both non-denaturing and SDS-PAGE. Immunoblotting after SDS-PAGE demonstrated labelling of peptides of 70-72, 28-32 and 16-18 kDa.     

Purification and characterization of heparin-binding growth factors from porcine uterus.

Heparin-binding growth factors present in pig uterine tissue were purified by approx. 50,000-fold using a combination of ammonium sulphate precipitation, ion-exchange chromatography and heparin-affinity chromatography. Purification of the uterus-derived growth factors (UDGFs) was monitored by the stimulation of [3H]thymidine incorporation into Swiss 3T3 cells and by a radioreceptor assay using 125I-labelled epidermal growth factor (EGF) as the ligand. The latter was shown to be a novel, rapid and reliable assay for heparin-binding growth factors which utilizes their trans-modulation of EGF receptor affinity. UDGFs exhibit strong affinity for immobilized heparin and two forms, named alpha UDGF and beta UDGF, were distinguished by salt gradient elution from heparin-agarose affinity columns. beta UDGF activity was eluted from heparin-agarose between 1.5 M- and 1.8 M-NaCl, and was correlated with the elution of a protein doublet of 17.2 kDa and 17.7 kDa. Immunoblotting of heparin-purified beta UDGF indicated that the beta UDGF doublet is immunologically related to the 146-amino-acid form of bovine basic fibroblast growth factor (bFGF), and that the 17.2 kDa component is an N-terminally truncated form of the 17.7 kDa component. After purification by C4 reversed-phase h.p.l.c., this doublet was biologically active and greater than 95% pure as assessed by silver-stained SDS/PAGE. Amino acid composition and sequence analysis confirmed that these beta UDGF polypeptides were microheterogeneous forms of bFGF. Fractions containing alpha UDGF activity were eluted from heparin-agarose in 1.3 M-NaCl. These fractions contained a 16.5 kDa protein which co-migrated on SDS/polyacrylamide gels with recombinant human acidic FGF (aFGF) and which which cross-reacted with an antiserum raised against aFGF. The identification of heparin-binding growth factors in porcine uterus at the time of implantation raises the possibility that they function in the reproductive tract during early pregnancy.