Nonequilibrium pH Gradient Gel Electrophoresis Research Articles

Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins

Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome.

Identification and Variation of Glutelin α Polypeptides in the Genus Oryza Assessed by Two-Dimensional Electrophoresis and Step-by-Step Immunodetection

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.

Human sperm protein encyclopedia and alloantigen index: mining novel allo-antigens using sera from ASA-positive infertile patients and vasectomized men

Anti-sperm antibodies (ASA) are an important cause of immunological infertility. The objective of this study was to identify immunodominant sperm antigens recognized by anti-sperm antibodies (ASA) in serum samples of infertile men, women and vasectomized men. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE), followed by PAGE and Western blotting. Serum samples from five infertile male and five infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT), were analyzed by Western blotting using NEPHGE gels followed by enhanced chemiluminescence (ECL) to identify the basic sperm antigens reactive to the sera. Serum samples from five fertile male and five fertile female subjects that were ASA-negative by IBT were used as controls. Serum samples from six vasectomized men collected before vasectomy and at different time intervals until 6 months after vasectomy were analyzed by Western blotting using IEF gels. The ECL blots were analyzed to compare immunoreactivity between serum samples from fertile and infertile subjects and identify antigens unique to sera of the infertile subjects. Similarly, immunoreactivity between serum samples from pre- and post-vasectomy was compared to identify antigens unique to sera collected following vasectomy. Five allo-antigenic basic protein spots were recognized by sera from infertile males but not from fertile subjects. Five sperm iso-antigenic basic spots were recognized by infertile female subjects. Two among six of the vasectomized men's sera showed a difference in the Western blot profile 6 months after vasectomy, recognizing at least one new protein spot in each case when compared to pre-vasectomy sera. The acrosomal protein SP-10 was identified as an alloantigen recognized by a post-vasectomy serum. Molecular identities of the known allo- and iso-antigens identified in this study and in previous studies from this laboratory are reviewed and discussed.

Calponin phosphorylation in cerebral cortex microvessels mediates sustained vasoconstriction after brain trauma

Objectives: The purpose of this study was to determine the molecular and biochemical changes in the contractile protein, calponin (Cp), which temporally coincide with a previously reported state of sustained contractility following traumatic brain injury (TBI).Methods: Double immunofluorescence, western analysis and two-dimensional non-equilibrium pH gradient gel electrophoresis (NEPHGE)/SDS-PAGE techniques were utilized to determine both the location and extent of Cp within smooth muscle cells (SM) and the phosphorylation state of Cp following TBI, as induced using a weight drop acceleration impact model.Results: Double immunofluorescence for Cp and SM indicate that following injury, Cp migrates from the cytosol to a location subjacent to the SM membrane. Western analysis revealed a significant increase in Cp protein expression following injury that was maintained up to 48 hours post-injury. Combined Western analysis and NEPHGE indicated that Cp is phosphorylated following TBI.Discussion: Cp migration and phosphorylation may underlie the mechanism for increased vasoreactivity leading to hypoperfusion following TBI.

Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells.

Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.

Monoclonal antibody DS6 detects a tumor-associated sialoglycotope expressed on human serous ovarian carcinomas.

A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium. CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes. Pre-treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase-sensitive and periodic acid-sensitive sialic acid glycoconjugate ("sialoglycotope"). SDS-PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non-disulfide-linked glycoprotein containing N-linked oligosaccharides. Two-dimensional non-equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5. Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double-label, 2-color immunohistochemical studies. The DS6-detected CA6 antigen appears to be distinct from other well-characterized tumor-associated antigens, including MUC1, CA125 and the histo-blood group-related antigens sLea, sLex and sTn.

High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.

Two-dimensional gel electrophoresis has been the technique of choice for analyzing the protein composition of cell types, tissues and fluids and is a key technology in modern proteomics projects. Here we describe reproducible procedures for running isoelectric focusing and nonequilibrium pH gradient electrophoresis gels that are based on the carrier ampholyte technology originally described by O'Farrell. Moreover, we present a sensitive immunoblotting procedure that has been used routinely in our laboratory to determine the identity of hundreds of human proteins.

Effects of environmental pH on membrane proteins in Borrelia burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, alternates between the microenvironments of the tick vector, Ixodes scapularis, and a mammalian host. The environmental conditions the spirochete encounters during its infectious cycle are suspected to differ greatly in many aspects, including available nutrients, temperature, and pH. Here we identify alterations in the membrane protein profile, as determined by immunoblotting and two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), that occur in virulent B. burgdorferi B31 as the pH of the medium is altered. Initial comparisons of cultures incubated at pHs 6.0, 7.0, and 8.0 yielded alterations in the expression of seven membrane proteins as determined by probing with hyperimmune rabbit serum. Six of these membrane proteins (54, 45, 44, 43, 35, and 24 kDa) were either present in increased amounts in or solely expressed by cultures incubated at pHs 6.0 and 7.0. The 24-kDa protein that decreased in expression at pH 8.0 was identified as outer surface protein C (OspC). In addition, a 42-kDa membrane protein increased in amount in cultures incubated at pH 8.0. Similar changes were observed with serum from a mouse infected by tick bite, with the recognition of two additional bands (48 and 46 kDa) unique to pHs 6. 0 and 7.0. When membrane fractions were analyzed by 2D-NEPHGE, at least 37 changes in the membrane protein profile between cells incubated at pHs 6.0, 7.0, and 8.0 were observed by immunoblotting and silver staining. Environmental cues such as pH may prove important in the regulation of virulence determinants and factors necessary for the adaptation of B. burgdorferi to the tick or mammalian microcosm.

Protein kinase C--catalyzed calponin phosphorylation in swine carotid arterial homogenate.

Calponin, a thin filament-associated protein, inhibits actin-activated myosin ATPase activity, and this inhibition is reversed by phosphorylation. Calponin phosphorylation by protein kinase C and Ca2+/calmodulin-dependent protein kinase II has been shown in purified protein systems but has been difficult to demonstrate in more physiological preparations. We have previously shown that calponin is phosphorylated in a cell-free homogenate of swine carotid artery. The goal of this study was to determine whether protein kinase C and/or Ca2+/calmodulin-dependent protein kinase II catalyzes calponin phosphorylation. Ca2+-dependent calponin phosphorylation was not inhibited by calmodulin antagonists. In contrast, both Ca2+- and phorbol dibutyrate/1-oleoyl-2-acetyl-sn-glycerol dependent calponin phosphorylation were inhibited by the pseudosubstrate inhibitor of protein kinase C and staurosporine. Our results also demonstrate that stimulation with either Ca2+, phorbol dibutyrate, or 1-oleoyl-2-acetyl-sn-glycerol activates endogenous protein kinase C. We interpret our results as clearly demonstrating that the physiological kinase for calponin phosphorylation is protein kinase C and not Ca2+/calmodulin-dependent protein kinase II. We also present data showing that the direct measurement of 32P incorporation into calponin and the indirect measurement of calponin phosphorylation using nonequilibrium pH gradient gel electrophoresis provide similar quantitative values of calponin phosphorylation.

Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells

In this study, we report the detection of a new group of five stress-inducible basic small heat shock proteins (BShsps) in Xenopus laevis kidney epithelial A6 cells by means of two-dimensional non-equilibrium pH gradient gel electrophoresis. These basic 30-kDa small hsps are distinct from the previously described X. laevis acidic hsp30 family on the basis of their charge and lack of cross-reactivity with an hsp30 antibody. Furthermore, at least two of the five BShsps were present constitutively, an observation that has not been made with the acidic hsp30 family. The heat inducibility of the BShsps was regulated at the level of transcription as indicated by their inhibited synthesis in the presence of the transcriptional inhibitor actinomycin D. Furthermore, the optimal temperature of BShsp induction, temporal pattern of synthesis, and induction of BShsps by other stressors such as herbimycin A and sodium arsenite were similar to those reported for the acidic hsp30 family. This study suggests that X. laevis contains at least two unique groups of small heat shock proteins that are coordinately expressed.Key words: Xenopus laevis, heat shock protein, hsp30, NEPHGE, basic small heat shock proteins, sodium arsenite, herbimycin A.

Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells.

In this study, we report the detection of a new group of five stress-inducible basic small heat shock proteins (BShsps) in Xenopus laevis kidney epithelial A6 cells by means of two-dimensional non-equilibrium pH gradient gel electrophoresis. These basic 30-kDa small hsps are distinct from the previously described X. laevis acidic hsp30 family on the basis of their charge and lack of cross-reactivity with an hsp30 antibody. Furthermore, at least two of the five BShsps were present constitutively, an observation that has not been made with the acidic hsp30 family. The heat inducibility of the BShsps was regulated at the level of transcription as indicated by their inhibited synthesis in the presence of the transcriptional inhibitor actinomycin D. Furthermore, the optimal temperature of BShsp induction, temporal pattern of synthesis, and induction of BShsps by other stressors such as herbimycin A and sodium arsenite were similar to those reported for the acidic hsp30 family. This study suggests that X. laevis contains at least two unique groups of small heat shock proteins that are coordinately expressed.

Phosphorylation of calponin in airway smooth muscle.

Calponin is an actin-binding protein known to be a substrate in vitro for several protein kinases and phosphoprotein phosphatases. We tested the hypothesis that calponin is phosphorylated in vivo using canine tracheal smooth muscle strips metabolically labeled with 32Pi. Calponin was gel purified from muscles stimulated with 1 microM carbachol. Phosphorylation increased to 2.0 times the basal level of 178 +/- 26 counts per minute (cpm)/microgram calponin within 30 s to 350 +/- 64 cpm/micrograms. Two-dimensional nonequilibrium pH gradient gel electrophoresis resolved four charge isoforms of calponin in unstimulated muscle. Stimulation with carbachol induced an additional more acidic isoform. Phosphorylation of calponin in vitro with protein kinase C (PKC) also induced formation of additional acidic isoforms. The functional effect of phosphorylation was demonstrated using an in vitro motility assay in which unphosphorylated calponin (2 microM) caused a profound inhibition of actin sliding. Calponin phosphorylated by PKC did not inhibit actin sliding. The results show that phosphorylation of calponin occurs in intact tracheal smooth muscle and that phosphorylation of calponin in vitro alleviates the inhibitory effect of calponin on actomyosin function.

Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database.

Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the beta-cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2-D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%-36.1%. When the same sample was analyzed repeatedly in one set of gels (intra-assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL-1beta.

Crystal Violet as an Indicator Dye for Nonequilibrium pH Gradient Electrophoresis (NEpHGE)

I found that a blue-colored dye, crystal violet, is a useful indicator for nonequilibrium pH gradient electrophoresis (NEpHGE). Unlike isoelectrofocusing, NEpHGE basically depends on the nonequilibrium gradient; therefore, the location of the proteins differs with acrylamide concentration or voltage used. To get reproducible results, colored marker which indicates the end of the electrophoresis might be very helpful. Crystal violet is an adequate dye for this purpose. This deeply colored, nontoxic material has a positive charge and migrated slightly faster than almost all proteins on NEpHGE. This dye affected neither the formation of pH gradient nor migration of proteins. Moreover, the ratio of migration (protein/dye) in the NEpHGE gels did not change under different conditions tested. Therefore, the addition of trace amounts of this dye gives a convenient endogenous color indicator to determine the end of the NEpHGE. Other positively charged dyes could be used as such an indicator, but some dyes (for example, methylene blue) lost their color by reduction during electrophoresis. Some dyes (for example, ethidium bromide) migrate too fast; therefore, they were not suitable indicators for NEpHGE.

Substitution of an aspartic acid for glycine 700 in the alpha 2(I) chain of type I collagen in a recurrent lethal type II osteogenesis imperfecta dramatically affects the mineralization of bone.

We describe a new dominant mutation of type I collagen responsible for a recurrent lethal osteogenesis imperfecta. Dermal cultured fibroblasts of the proband produced both normal and overmodified type I collagen chains. Previous results (Cohen-Solal, L., Bonaventure, J., and Maroteaux, P. (1991) Hum. Genet. 87, 297-301) and cyanogen bromide peptide mapping after non-equilibrium pH gradient gel electrophoresis indicated that the anomaly was a charge mutation localized in the alpha 2CB3-5A. The mutation was identified as a G to A transition in the COL1A2 gene, which converts glycine 700 to aspartic acid in the alpha 2I chain. This mutation caused the abolition of a ScrFI site, which was also absent in the suspected mosaic father. Pulse-chase experiment showed intracellular retention and increase of the degradation of the synthesized collagen. To understand more directly the tissue defect in osteogenesis imperfecta, skin and especially bone were studied with biochemical and transmission electron microscopy techniques. Collagen matrix of both tissues was dramatically decreased and presented a retarded migration, showing that abnormal molecules were incorporated during the fibrillogenesis. The abnormal collagen mostly remained within the fibroblasts and osteoblasts, which presented typical features of intracellular retention. We observed the presence of spheritic aggregates of mineral, unrelated to the scarce and thin collagen fibrils, in bone. Such abnormal mineralization could be the consequence not only of the decrease of the collagen content but more importantly of the inability of the abnormal molecules to form an organized network necessary to the deposition of apatite crystallites.

Identification of bacteriophage T4 prereplicative proteins on two-dimensional polyacrylamide gels.

Bacteriophage T4 makes a large number of prereplicative proteins, which are involved in directing the transition from host to phage functions, in producing the new T4 DNA, and in regulating transcriptional shifts. We have used two-dimensional gel electrophoresis (nonequilibrium pH gradient electrophoresis gels in the first dimension and sodium dodecyl sulfate-polyacrylamide gradient slab gels in the second) to identify a number of new prereplicative proteins. The products of many known genes are identified because they are missing in mutants with amber mutations of those genes, as analyzed by us and/or by previous workers. Some have also been identified by running purified proteins as markers on gels with labeled extracts from infected cells. Other proteins that are otherwise unknown are characterized as missing in infections with phage carrying certain large deletions and, in some cases, are correlated with sequence data.

Calponin phosphorylation in vitro and in intact muscle.

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.

Plasma membrane p180, which insulin receptor phosphorylates in vivo, is not a tyrosine kinase

The earliest substrates to the transmembrane insulin receptor tyrosine kinase, that would function in insulin signalling, are likely to be associated with the plasma membrane. Rat liver plasma membrane 180, 000 M r protein (p180) is a substrate to the insulin receptor in vitro [Goren et al. (1990) Cellular Signalling 2, 537–555]. The question as to whether p180 is a substrate in vivo was addressed. Half ml 0.9% NaCl or 500 μg insulin was injected into rat livers. Purified plasma membrane glycoproteins from the livers were assayed for in vitro phosphorylation reaction products and endogenous tyrosine-phosphorylated proteins. Membranes from insulin-injected rat livers contained phosphorylated p180 and phosphyrylated insulin receptor β-subunit, whereas saline-injected rat liver membranes contained neither. These data suggested that p180 is an in vivo substrate to the insulin receptor. In vitro p180 is tyrosine-phosphorylated in the absence of insulin. p180, therefore, may be the epidermal growth factor (EGF) receptor or another tyrosine kinase that could be part of a phosphorylation cascade initiated by insulin. Two different experiments suggested that p180 is not the EGF receptor: (i) two-dimensional gel electrophoresis (first dimension—non-equilibrium pH-gradient gel electrophoresis) indicated that p180 is a more basic glycoprotein than EGF receptor; and (ii) based on reverse-phase high pressure liquid chromatography, the tryptic-phosphopeptides of carboxymethyl-Sepharose-purified phosphorylated-p180 were different from those of A431 cell phosphorylated-EGF receptor. Similarly, two different experiments demonstrated that p180 is not a tyrosine kinase: (i) gel-permeation chromatogrpahy separated the insulin receptor from p180 and only insuling receptor was autophosphorylated in vitro; and (ii) membrane proteins not bound to immobilized ATP contained p180. Thus, p180 can associate with the insulin receptor and be phosphorylated in vitro and in vivo; however, p180 does not function in an insulin receptor-mediated phosphorylation cascade.

Isolation and characterization of three families of auxin down-regulated cDNA clones

Five cDNA clones (ADR6, ADR11-1, ADR11-2, ADR12-1 and ADR12-2), representing three families of auxin down-regulated (ADR) genes were isolated and characterized. These were isolated by screening a lambda Zap cDNA library with the partial cDNA clones p6, p11 and p12, isolated earlier (Baulcombe and Key, J Biol Chem 255: 8907-8913, 1980). Hybrid-select translation of ADR6, ADR11-2 and ADR12-2 clones produced polypeptides of 33 kDa 22.5 kDa and a 6 and 7 kDa respectively, when analyzed by SDS-PAGE. ADR6 and ADR12-2 gave one and two spots, respectively, on an IEF-SDS 2D gel. ADR11-2 probably encodes a basic protein as it was only resolved on non-equilibrium pH gradient gel electrophoresis (NEPHGE). Genomic Southern blot analysis of ADR6, ADR11 and ADR12 suggests that each represents a small multigene family. The RNA levels corresponding to ADR6, ADR11 and ADR12 decrease in response to applied auxin by 100-, 15- and 10-fold, respectively (Baulcombe and Key, 1980). Runoff transcription, done in the presence and absence of auxin, showed that the rate of transcription of the genes corresponding to ADR6, ADR11-2 and ADR12-2 was reduced in the presence of auxin, but the decrease was small relative to the decrease in the cytoplasmic levels of these mRNAs, in response to auxin. A comparative analysis of the influence of auxin on in vitro transcription and steady state RNA levels corresponding to these ADR cDNAs suggests that the decrease in rate of transcription due to auxin is not enough to account for the auxin-induced decrease in the steady state levels. Northern analysis showed developmental and organ/tissue-specific response of these ADR genes. Furthermore, the expression of the genes corresponding to ADR6 and ADR12-1 appears to be up-regulated by light, whereas the gene corresponding to ADR11 appears to be down-regulated by light.

Phosphorylation, calpain proteolysis and tubulin binding of recombinant human tau isoforms

In this study, the phosphorylation, calpain hydrolysis and tubulin binding of three recombinant human tau isoforms were examined. The three isoforms used in these studies were tau with three (T3) or four (T4) tandemly repeated tubulin binding domains located in the carboxy-terminal half of the molecule; and tau with four-tandem repeats and a 58-amino acid insert in the amino terminus (T4L). Both cAMP-dependent protein kinase (cAMP-PK) and Ca 2+ / calmodulin-dependent protein kinase II (CaMKII) readily phosphorylated the three human tau isoforms, although cAMP-PK phosphorylated them to a significantly greater extent than CaMKII. Phosphorylation of T3, T4 and T4L by cAMP-PK or CaMKII resulted in the slowed migration of the protein bands on sodium dodecyl sulfate-polyacrylamide gels and a shift of the isoelectric variants to more acidic positions on two-dimensional non-equilibrium pH gradient electrophoresis gels compared with controls. However, the phosphorylation-induced changes in the electrophoretic migration of the tau isoforms were unique for each kinase. Two-dimensional phosphopeptide maps and sequential phosphorylation experiments indicate that cAMP-PK phosphorylates sites in the human tau isoforms that are phosphorylated by CaMKII, as well as unique sites that are not phosphorylated by CaMKII. T3, T4 and T4L were hydrolyzed similarly by calpain; however, the calpain proteolysis of the recombinant tau isoforms was significantly faster than the proteolysis of human or bovine tau. Phosphorylation of the isoforms by either cAMP-PK or CaMKII did not alter the rate or extent of calpain proteolysis. Phosphorylation of T3, T4 and T4L by cAMP-PK decreased isoform-tubulin binding compared to controls, however only the results with T3 were statistically significant.