Acidic Isoelectric Point Research Articles

Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5′-monophosphate-dependent protein kinase

We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832–838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098–2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII α and RII β isoforms. An RII β monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII α RII β monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII α RII β monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[ 32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[ 32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 M r fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).

Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for Western blot analysis

Western blotting of low molecular weight ( M r) acidic and basic isoelectric point (p I) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, M r 6045, p I 4.4), transforming growth factor type α (TGFα, M r 5623, p I 6.8), insulin-like growth factor (IGF-I, M r 7649, p I 8.3), and basic fibroblast growth factor (bFGF, M r 15,000–17,000, p I 9.6) all transferred with high efficiency (74.1 ± 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46–98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% ( v v ) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGFα, and IGF-I were not detectable while 6.25–100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic p I proteins.

A developmentally regulated chicken neuronal protein associated with the cortical cytoskeleton

Monoclonal antibody 3D5 recognizes a single component of the neuronal membrane skeleton isolated from the chicken embryo brain. The 3D5 antigen is highly enriched in the CNS, and smaller amounts are found in the PNS. It is also present in non-neural tissues, but this is due to peripheral innervation. The biochemical and molecular properties of the 3D5 antigen are very similar to those of the previously described mammalian protein B-50 (Zwiers et al., 1985)/GAP 43 (Jacobson et al., 1986)/pp46 (Ellis et al., 1985)/F1 (Chan et al., 1986), and include anomalous SDS gel migration, acidic isoelectric point, and extraction from the cytoplasmic side of the plasma membrane only under extremely alkaline conditions. The 3D5 antigen is also developmentally regulated, with maximum expression in brain occurring at E14-E16, after which levels decrease approximately 4-fold in the adult. Immunofluorescence staining of cultured neurons shows that the 3D5 antigen is located in all parts of the cell but is particularly enriched in the growth cone and the growth cone filopodia. As the 3D5 antigen is enriched in the membrane skeleton, we suggest that this protein is involved in an association between the actin-containing cytoskeleton and the plasma membrane.

Genetic studies of low-abundance human plasma proteins. XII. A new variant of corticosteroid-binding globulin detected by isoelectric focusing/immunoblotting.

Thin-layer polyacrylamide gel isoelectric focusing over the pH range 3.5-5 followed by immunoblotting was used to investigate the occurrence and frequency of genetic variation in corticosteroid-binding globulin (CBG). Plasma samples from US Caucasians (n = 105) and US Blacks (n = 106) from Pittsburgh, Pa., Canadian Indians from Vancouver Island (n = 91) and Nigerian Blacks (n = 116) were analyzed. A complex isoprotein pattern was observed in all individuals tested. Reduction of this pattern to a single primary band following neuraminidase treatment indicates that the observed intraindividual variation is due to variation in the number of sialic acid residues associated with CBG. The CBG variant pattern consisted of a series of isoprotein bands having the same mobility as the common pattern, and a second series of bands at a more acidic isoelectric point. This pattern is consistent with heterozygosity for a rare CBG allele.

Tropomyosin in the sea urchin egg cortex

Tropomyosin was purified from the Triton-treated cortex fraction of fertilized sea urchin egg. Egg tropomyosin showed characteristics typical of nonmuscle tropomyosins such as low molecular mass, short periodicity of Mg2+-paracrystals, low lysine/arginine ratio, high Mg2+ requirement in binding to F-actin, in addition to the properties of all tropomyosins, namely, stability to high temperature, anomalous migration of SDS/urea gel, dissociation from F-actin under high ionic conditions and very acidic isoelectric point. Co-sedimentation assay of egg tropomyosin with actin in the presence of the previously purified high-molecular-mass actin binding protein (260-kDa protein) showed that these two proteins bind to actin filaments in a non-competitive manner. This suggested that both the proteins play a cooperative role in the formation of actin-filament-based cytoskeletal structure in the cortex.

Biochemical characterization of membrane cofactor protein of the complement system.

Membrane cofactor protein (MCP; formerly termed glycoprotein 45-70 to indicate its Mr) of complement is a widely distributed iC3/C3b binding protein with co-factor activity. On human mononuclear cells and cell lines and platelets, MCP is a doublet. The two forms differ in Mr by approximately 5 k and the upper species is predominant in most individuals. To further characterize these two forms, limited proteolytic digestions were performed. Of the four peptides produced, three have identical Mr indicating that the molecules are similar proteins. Both forms also have acidic isoelectric points and shift to a less acidic isoelectric point after treatment with neuraminidase. Glycosidase digestions indicate that both species contain N- and O-linked oligosaccharides but that the quantity of sialic acid is greater on the larger one. Pulse-chase experiments demonstrate approximately equal quantities of two precursor forms with Mr of 41 and 43 k. These two precursors possess N-linked high-mannose type of oligosaccharides and chase into the mature molecules which have complex sugars. The smaller precursor chases at a slower rate, possibly accounting for the reduced quantity of the smaller form of the mature form of MCP. These experiments indicate that the two forms of MCP are structurally similar and are derived from two distinct precursors. They also suggest that variations in the rate of processing of two intracellular precursors may account for the different quantities of the mature forms of this membrane protein.

Alteration of a developmentally regulated, heat-stable polypeptide in the lens of the Philly mouse. Implications for cataract formation.

The beta-crystallin basic principal polypeptide (beta Bp) appears to be altered in the lens of the Philly mouse and may be the main defect in this hereditary cataract. Northern blot analysis showed that an mRNA encoding for beta Bp is present in the Philly mouse lens, but normal beta Bp could not be detected. Instead, a different protein related to beta Bp has been observed. Western blot analysis with antibodies against specific beta Bp peptide sequences showed that the Philly protein shares the same amino-terminal residue as beta Bp but lacks a part of the carboxyl-terminal half of normal beta Bp. The altered protein is slightly smaller than beta Bp and has a more acidic isoelectric point by two-dimensional gel electrophoresis. It also lacks the property of heat stability characteristic of normal beta Bp. The mapping of the alteration in beta Bp may give insight into the nature of the heat stability of this protein as well as some indication of the structural components that are necessary to maintain optical clarity in the lens.

CDNA cloning and sequence of a new type of actin in mouse B16 melanoma.

Parent B16 melanoma and B16-F1 cell lines express a third actin (Ax) in addition to beta- and gamma-actin. It has the same molecular mass (43,000 daltons) and a more acidic isoelectric point (pI = 5.2) than the latter two actins (pI = 5.3) (Taniguchi, S., Kawano, T., Kakunaga, T., and Baba, T. (1986) J. Biol. Chem. 261, 6100-6106). We constructed a cDNA library from poly(A)+ RNA of B16-F1 and then isolated Ax actin candidate clones. According to the nucleotide sequencing analysis for one of the candidate clones, pMA 30, the predicted amino acid sequence was composed of 375 amino acids and was similar to that of beta-actin, but differed at the 28th amino acid in that leucine replaced the arginine of beta-actin. When RNA synthesized from the clone pMA 30 with the SP6 transcription system was translated in vitro using reticulocyte lysate, we identified a polypeptide which had the same isoelectric point and molecular weight as Ax actin; the polypeptide had binding activity to DNase I, a common characteristic of native actin. These observations provide evidence that the clone pMA 30 encodes the mRNA for Ax actin. In the nucleotide sequence of the Ax cDNA, there are: 1) one base change in the coding region which causes a loss of the SmaI site and an amino acid exchange, as mentioned above; 2) four deletion sites in the 3'-noncoding region; 3) one insertion site in the 3'-noncoding region; and 4) one base change in the 5'-noncoding region, as compared with hitherto known mouse beta-actin cDNA. These differences between Ax and beta-actin cDNA indicate that the Ax actin is encoded by an unique gene set, independent of beta-actin.

Expression in Escherichia coli of full-length and mutant rat brain calbindin D28. Comparison with the purified native protein.

Studies of vitamin D-dependent 28-kilodalton calcium binding protein (calbindin D28) have been hindered by difficulties in purifying large amounts of the protein. In order to overcome this problem, we cloned and expressed a full-length rat brain calbindin D28 cDNA. In addition, we isolated and purified to homogeneity, native rat brain calbindin D28. The isolated native protein has an apparent molecular mass of 27 kDa and properties similar to those of the well-characterized chicken calbindin D28. It has an acidic isoelectric point (approximately 4.5), a high affinity for calcium, and an amino terminus blocked to Edman degradation. The properties of the native and the recombinant proteins were examined by gel electrophoresis, isoelectric focusing, protein sequencing, amino acid composition analysis, and calcium binding assays. We demonstrated that: (i) the authentic and the full-length recombinant proteins have similar molecular weights and isoelectric points; (ii) the proteins have the same amino acid composition; (iii) the proteins bind calcium in a similar manner; (iv) the absence of a blocking NH2-terminal group in the recombinant protein does not appreciably influence the binding of calcium. To further examine the calcium binding properties of this protein, we constructed deletion mutants lacking one or both of the two putative degenerated calcium binding sites (EF hand regions). These deletions resulted in smaller proteins that still bound calcium. The ability to express and purify calbindin D28 and mutants thereof should allow the systematic elucidation of structure-function relationships in this class of calcium binding proteins.

Biochemical properties of glycoproteins involved in lymphocyte recognition of high endothelial venules in man.

Lymphocyte interactions with high endothelial venules (HEV) are important to the in vivo migration of normal and neoplastic lymphocyte populations. We have previously described an 85- to 95-kDa lymphocyte surface glycoprotein(s) defined by mAb Hermes-1, that is involved in the recognition of HEV by human lymphocytes: antibodies against distinct epitopes of the Hermes-1 Ag differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial HEV. Here we characterize further the Hermes-1-defined glycoproteins. No well defined differences were observed between the Hermes-1 Ag immunoprecipitated from PBL and from mucosa- vs lymph HEV-specific cell lines. The Ag is an acidic (isoelectric point = 4.2) sulfated molecule bearing both O-linked and (3,4) N-linked oligosaccharide side chains. A subset of the Hermes-1-immunoprecipitated species is modified by covalent linkage to chondroitin sulfate, yielding a Mr of approximately 180 to 200 kDa. Pulse-chase labeling reveals a major precursor of 76 kDa that appears to be processed either to the 85- to 95-kDa form or, by addition of chondroitin sulfate, to a 180- to 200-kDa form. The potential role of these structural modifications, and particularly of chondroitin sulfate, in the function of the putative adhesion molecules is discussed.

Characterization of two highly phosphorylated cytoskeleton-associated proteins, pp58 and pp60, in tumoricidal murine peritoneal macrophages and their comparison with vimentin

Two TX-insoluble cytoskeleton-associated proteins, pp58 and pp60, become highly phosphorylated in tumoricidal murine peritoneal macrophages. Results suggest that pp58 (pI 5.00) is phosphovimentin because it is highly insoluble in TX, shares the same mol. wt as vimentin, has a more acidic isoelectric point than vimentin, is phosphorylated primarily at serine, and generates the same V-8 protease peptide map as vimentin. pp60 generates at slightly different peptide map than pp58 and has a slightly less acidic isoelectric point (pI 5.02) than pp58 (pI 5.00), but is similar to pp58 by being highly insoluble in TX and being phosphorylated primarily at serine residues. Pulse-chase experiments demonstrate that pp58 is not a precursor to or breakdown product of pp60, or vice versa because they show similar rates of [ 32P]-phosphate incorporation and turnover.

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor).

HPRT Ann Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in wo brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased Km for both substrates, a normal Vmax, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT Ann Arbor and determined its complete nucleotide sequence. A single nucleotide change (T----G) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5'-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.

Protein synthesis in human platelets

The platelets and leukocytes of human peripheral blood were separated and cultured for 24 h in the same medium. The culture medium was completed with S-methionine. The overall protein synthetic activity of the platelets, compared with leukocytes, was studied with a two-dimensional gel electrophoresis technique combined with fluorographic detection of labelled proteins. Our findings confirm the platelet-origin of a few newly synthesized proteins. The most pronounced newly synthesized platelet protein with a molecular weight of approximately 35 Kda and with a more acidic isoelectric point than that of actin was identified as a cytoskeleton-associated protein.

Comparison of two lignin-degrading fungi:Phlebia radiata andPhanerochaete chrysosporium

The ligninolytic enzymes ofPhlebia radiata were produced in static conditions earlier developed forPhanerochaete chrysosporium. The production pattern of lignin peroxidases resembled that ofP. chrysosporium. The extracellular proteins ofPhlebia radiata were separated by isoelectric focusing. Four proteins with acidic isoelectric points (≤4.15) were detected by peroxidase staining. The peroxidases ofP. radiata reacted with antibodies produced against a peroxidase ofPhanerochaete chrysosporium and vice versa. Thus the lignin peroxidases of the two fungi have major similarities despite slight differences in their isoelectric points and molecular weights. Veratryl alcohol was produced by both fungi and degraded to veratraldehyde, two lactones and a quinone by the ligninolytic cultures.

Biochemical differentiation of clones of OcaOxalis tuberosa, Oxalidaceae) by Their tuber proteins and the properties of these proteins

Tuber proteins of oca (Oxalis tuberosa) were separated for clone (cultivar) differentiation by gel electrophoresis in polyacrylamide. The patterns of native proteins and especially of esterases in a porosity gradient with and without urea were suitable for differentiation, whereas SDS-electrophoresis of protomers or focusing of native proteins yielded more or less the same patterns for 23 different accessions. The main proteins are quite acidic (isoelectric points pH 4.7–4.9) and have a relative molecular weight of 17–18 KD (Kilo-Dalton). Amino acid analysis of the protein precipitate by propan-2-ol from tuber sap revealed proteins rich in aspartic and glutamic acid (one third of all amino acids) with a considerable share of essential ones.

Identification of multiple low molecular weight placental prolactin-like proteins produced by rat trophoblast cells

Rat trophoblast tissue was found to synthesize a number of low molecular weight proteins possessing prolactin-like characteristics. There appear to be at least three proteins that cross-react with antisera to pituitary prolactin. Two of the proteins had a molecular weight of 25,000, similar to ovine pituitary prolactin, and isoelectric points of 6.8 and 7.0. The third immunoreactive protein had a lower molecular weight (23,500), similar in size to human placental lactogen, and a slightly more acidic isoelectric point of 6.75. The molecular weight variants cross-reacted with an antipeptide serum that was generated to a synthetic peptide representing amino acids 150 to 164 of rat placental lactogen-2 (PL-2). Based on this analysis, we consider these proteins to be related to PL-2. Analysis of trophoblast proteins by gel-filtration chromatography resulted in the identification of another trophoblast prolactin. This material eluted earlier than PL-2-related proteins on a gel-filtration column, possessed prolactin-like activity (determined by competition with ovine pituitary prolactin for rabbit mammary gland or rat liver prolactin receptors) but showed limited cross-reactivity with either the antiserum to pituitary prolactin or the antiserum to the PL-2 peptide. We have thus identified multiple low molecular weight trophoblast prolactins, possessing different biochemical and immunological characteristics.

The concentration of bovine placental lactogen and the incidence of different forms in fetal cotyledons and in fetal serum

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9±0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean ± S.E.M.) in amniotic and allantoic fluid was 0.4±0.1 and 1.2±0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.

The Partial Dependency of Human Prostatic Growth Factor on Steroid Hormones in Stimulating Thymidine Incorporation into Dna

Growth factors stimulating DNA synthesis in mouse 3T3, human DU145, and LNCaP were partially purified from human benign hyperplastic and cancerous prostates. These factors have a high affinity for heparin sepharose and are eluted from the heparin-sepharose column, at 1.2 to 1.9 M NaCl. In normal prostates, the high affinity human prostatic growth factor occurred only in extremely small amounts. The high affinity growth factors stimulate DNA synthesis in 3T3, DU145, and LNCaP. Stimulation was significantly enhanced by 5α-dihydrotestosterone and 17β-estradiol in the androgen sensitive LNCaP cell line. SDS-PAGE and isoelectrofocusing confirmed that the partially purified factors had a molecular weight of 18kDa and acidic isoelectric points of pH 3.6 and 4.7.

Complexity of cellulases from Trichoderma reesei with acidic isoelectric points: A two-dimensional gel electrophoretic study using immunoblotting

Antibody specific to Trichoderma reesei cellulase (65 kDa, isoelectric point, pI, 7.7) shows immuno-cross reactivity with acidic hydrolase complexes containing other cellulases, (pIapp. 3.4–4.5) when tested under conditions of 2D-electrophoresis (1st dim. PAGIF, 2nd dim. SDS-PAGE) together with Western blotting. Degradation pattern of 14C(U)-labeled G1–G5 of the 65 kDa cellulase was followed by a 2-directional oligodextrin mapping procedure. Using preparative IEF, homologous antigen portions were detected in cellulases present within acidic hydrolase complexes showing mainly identical molar weight (Mr 65 kDa and 57 kDa) but a range of charge (pI 3.4–4.5). The pattern of acidic cellulases as found after analytical 2D-electrophoresis was reconstituted by preparative IEF (pIapp. 2.7–5.1) followed by SDS-PAGE separation. Homogeneous fractions (upon IEF) gave up to 8 different polypeptides per complex upon SDS-PAGE (Mr 70−20 kDa). Charge heterogeneity of individual acidic hydrolase complexes upon IEF is discussed as one reason for ‘multiplicity’ of acidic cellulases.

Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti-gp330 immune complexes form in epithelial coated pits and rapidly become attached to the glomerular basement membrane.

The nephritogenic antigen of Heymann's nephritis (HN), gp330, was previously demonstrated (4-9) to be a resident glycoprotein of coated pits in the glomerular and proximal tubule epithelium of rats, and anti-gp330 IgG given intravenously was found to form IDs in glomeruli (passive HN). The purpose of this study was to investigate the detailed events that occur in the formation of IDs in passive HN. HN was induced by the injection of either 125I-labeled or unlabeled anti-gp330 IgG. At various times after injection (15 min to 8 d) the kidneys of some of the injected rats were fixed by perfusion, and the distribution of the rabbit IgG was determined by immunofluorescence and by immunoelectron microscopy. Glomeruli were isolated from the kidneys of injected rats and used for isolation of GBM fractions or for elution of the bound IgG. At 15 min to 1 h after injection, the rabbit IgG was localized by immunocytochemistry exclusively in coated pits along the podocyte plasmalemma facing the GBM. By 1-8 d, anti-gp330 IgG was detected in larger electron-dense IDs often located under the slit diaphragms. Serial sectioning revealed that each of the IDs maintained contact with a coated pit at some level. When GBMs isolated from rats given radiolabeled anti-gp330 IgG were examined by electron microscopy, the IDs were found to remain attached to the GBMs as early as 15 min after injection and coisolated with them at all time points. By double-immunolabeling of the isolated GBMs with two sizes of gold particles, both the antigen (gp330) and the anti-gp330 IgG could be demonstrated in IDs at all time points. When the amount of radiolabeled anti-gp330 bound to GBM fractions was compared with that of isolated glomeruli, it was found that 20% of the radiolabel remained bound to the purified GBMs at 15 min after injection, and 90% at 3 d. The bound IgG was released only by treatments that disrupt antibody-antigen complexes (high and low pH), but not by the other treatments we tried (detergent, high salt, heparinase, or collagenase digestion). When the IgG bound to glomeruli was eluted with acid citrate buffer 3 d after injection, it was found to specifically immunoprecipitate only gp330 from detergent-solubilized 125I-labeled kidney microvillar vesicles. By isoelectric focusing the eluate was found to be enriched in IgGs with acidic isoelectric points.(ABSTRACT TRUNCATED AT 400 WORDS)