Isoelectric Focusing Patterns Research Articles

COMPARATIVE STUDY OF SOLUBLE GONAD PROTEIN FOR BOTH SEXES OF SIGANUS RIVUIATUS (FAMILY: SIGANIDAE) IN RELATION TO MATURITY STAGES BY ISOELECTRIC FOCUSING

Soluble gonad protein for both sexes of Siganus rivulatus was analysed by isoelectric focusing in seven maturity stages. Sex- specific electrophoretic and isoelectric focusing patterns were found. More protein fractions are identified for stage II of both sexes than for die other stages. The total number of protein bands for female was greater than those of male. In female, spawning stage has the maximum number (4) of specific band while in male the maximum ones (6) were separated from stage (HI). The number of common bands in both female and male was (3 & 2) respectively. Clear differences in protein patterns of the seven stages for both sexes can be used to characterize the breeding season.

Determination of the origin of charge heterogeneity in a murine monoclonal antibody.

The aim of this study was to elucidate the molecular basis of charge heterogeneity found in a purified monoclonal IgG1 antibody, MMA383. Cation exchange chromatography (CEX) and isoelectric focusing (IEF) were used to monitor charge heterogeneity. CEX in conjunction with carboxypeptidase B digests of the antibody was used to determine the contribution of C-terminal lysines to MMA383 charge heterogeneity. Potential chemical degradation sites were identified by peptide mapping of individual chains, with peptide identification by mass spectrometry (MALDI-TOF MS). Peptide sequencing was used to determine specific deamidation sites. Binding constants of predominant isoforms were compared by surface plasmon resonance (SPR). Extensive charge heterogeneity of purified MMA383 was detected by CEX and IEF. Removal of C-terminal lysines simplified the IEF pattern to nine predominant isoforms. Quantitation of isoaspartate in each of the isoforms indicated deamidation of MMA383 as a major cause of charge heterogeneity. CEX of the individual isoform chains suggested the presence of one deamidation site on each of the heavy and light chains. The two sites of deamidation were identified using peptide mapping, sequencing and mass spectrometry. SPR results showed no significant difference in the binding parameters among the isoforms. C-terminal lysine microheterogeneity and deamidation of Asn141 in the heavy chain and Asn161 in the light chain are the major causes of MMA383 charge heterogeneity. Identification of the two deamidation sites will allow replacement of these amino acids in order to create a product less susceptible to degradation.

Expression and partial purification of a recombinant secretory form of human liver carboxylesterase.

Serine-dependent carboxylesterases (EC 3.1.1.1) are found in a variety of tissues with high activity detected in the liver. Carboxylesterases (CaE) hydrolyze aliphatic and aromatic esters, and aromatic amides, and play an important role in the detoxification of xenobiotic chemicals that contain organophosphate (OP) compounds. The detoxifying ability of CaE is limited by its low concentration in serum where it encounters OP compounds. Studies in our laboratory have shown that a pRC/CMV-hCaE plasmid construct, stably integrated into 293T cells, expresses a human liver CaE in culture. However, the enzyme remained inside the cell and reached a low steady-state level of expression. The goals of this study were to overexpress a functional human liver CaE from a recombinant cDNA in a human cell line and to isolate and purify the recombinant protein. To accomplish these goals, a single amino acid change was made in the C-terminal retrieval signal, HIEL (His-Ile-Glu-Leu), of human liver CaE. The mutation produced a unique Eco47III restriction site, which aided in clone selection. The recombinant plasmid, pRc/CMV-mhCaE, was isolated and stably integrated into human 293T cells. Expression of the altered cDNA resulted in secretion of an active CaE up to levels of 500 enzyme units per liter of growth medium. Secretory CaE displayed isoelectric focusing patterns similar to those of the native enzyme with no observable changes in activity. The secreted enzyme was partially purified by hydrophobic interaction chromatography and Cibacron blue affinity chromatography. Partial enzyme purification was achieved, and CaE retained a high level of enzymatic activity.

New diagnosis and treatment of congenital hepatic fibrosis.

New diagnosis and treatment of congenital hepatic fibrosis.

In situ Detection of Human Monocyte/ Macrophage Serine Esterase-1 mRNA Expression in Human Tissues

Human monocyte/macrophage serine esterase (HMSE) can be distinguished from esterases of other blood cells by a specific isoelectric focusing pattern of five enzyme variants. Using a HMSE-1 cDNA, expression of mRNA was investigated by nonradioactive in situ hybridization of human biopsy specimens and cytospin preparations. HMSE-1 transcripts could be detected in the myelomonocytic cell line U-937, blood monocytes and in tissue macrophages. Immune accessory cells of the monocyte/macrophage lineage did not express detectable amounts of HMSE-1 mRNA. These results indicate that HMSE-1 represents a cell-lineage-specific enzyme which can be used to characterize physiological and neoplastic variants of the human monocyte/macrophage cell system. The method described will enable further insight into the physiological function of HMSE-1.

Soybean seed pectinesterase

AbstractMethanol accumulates in axis tissues of maturing soybean seeds, correlating with preharvest seed deterioration. Accumulation of methanol appears to be associated with the enzymic demethylation of pectin methyl esters by pectinesterase (PE; EC 3.1.1.11). To characterize PE in developing and maturing soybean (Glycine max (L.) Merrill) seeds, enzyme activity was assayed in axis and cotyledon tissues. Activity per g fresh weight was 20–25 times higher in axes than in cotyledons with highest activities between 45 and 60 days after flowering (DAF). Twenty to 33% of the total PE activity was in the ‘soluble’ form (extracted with water, 0.5 M sucrose, 1 M sucrose and water). Soluble and cell-wall-bound PE (subsequently extracted with 1 M NaCI) were purified and characterized from axes of seeds at 45–60 DAF. Purification of PE was achieved through concentration of extracts by ultra-filtration, precipitation with ammonium sulfate (30–80% saturation), dialysis, gel filtration on Sephadex G-75 columns, and ion exchange chromatography on CM Sepharose CL-6B. Further purification of both soluble and bound PE was by isoelectric focusing (IEF) on ultrathin layers of polyacrylamide gel with simultaneous detection of protein and PE activity. It was possible to follow seven bands exhibiting PE activity with pl values between 6.0 and 9.5 in 1 M NaCI-extracts of total homogenates. Differences in the IEF patterns of bound and soluble PE were observed. Whereas the bound enzyme exhibited more basic PE bands (pl 8–9.5), the soluble enzyme had more active bands at pl 6.5, 7.0 and 7.5. The Mr was close to 33 000 and the pH optimum was 7.8 for both soluble and bound PE.

Genetic variation of β‐amylase thermostability among varieties of barley, Hordeum vulgare L., and relation to malting quality

AbstractTo investigate variation in the thermostability of β‐amylase among varieties of barley, Hordeum vulgare L., crude enzyme was extracted from the seeds, and the relative remaining activity was calculated after heat treatment. Our results indicated that the varieties tested were divided into three groups (types A, B and C). All the latest Japanese malting varieties showed high themostability (type A), while European, North American and Australian varieties showed intermediate (type B) or low thermostability (type C). Isoelectric focusing (IEF) of β‐amylase was also investigated. Type B varieties divided into two subtypes (types B1 and B2) based on two different IEF patterns (types I and II), whereas those of types A and C showed only one IEF pattern (type II). We also examined their thermostability in the varieties based on the pedigrees of the Japanese malting barley. Our results indicated that the thermostability of β‐amylase had a close relationship to fermentability for the production of beer. This fact suggests that the thermostability of β‐amylase has a significant influence on the malting quality of barley.

P40phox Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase: IMPLICATION OF A PROTEIN KINASE C-TYPE KINASE IN THE PHOSPHORYLATION PROCESS

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67(phox) (phagocyte oxidase), p47(phox), and p40(phox), which translocate to the membrane upon activation. In a previous paper, we reported that p40(phox) undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531-539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40(phox), we show that p40(phox) is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40(phox) in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40(phox). Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40(phox) are phosphorylated during activation of the NADPH oxidase.

Disease-related variations of the glycosylation of haptoglobin in the dog

Haptoglobin phenotypes have been shown in human medicine to be related to the prevalence of various diseases. Furthermore, abnormal glycosylation of haptoglobin has been reported as a consequence of liver disease, cancer and immunological disorders in man. To our knowledge, similar findings have not, so far, been reported in canine disease. The present paper describes a method for investigation of canine haptoglobin phenotypes and of microheterogeneity caused by altered glycosylation. The method consisted of isoelectric focusing (IEF) of dog serum, followed by immunoblotting. The results indicated the existence of only one canine haptoglobin phenotype with a characteristic microheterogeneity pattern in healthy dogs. Changes in this pattern were found in serum from dogs with liver disease, predominantly chronic progressive hepatitis, and with different kinds of anaemia. Pretreatment of serum with neuraminidase or glycopeptidase F (PNGase F) resulted in identical IEF patterns of haptoglobin from healthy and diseased dogs. Moreover, a fucose-specific lectin was capable of binding to some of the abnormal haptoglobin fractions, mainly those found in association with anaemia. The changes described were interpreted as alterations of the carbohydrate content, with or without fucosylation, of some haptoglobin fractions.

Identification of stathmin-like proteins in plants

Stathmin-like proteins were identified in broad bean, green bean, pea, soybean, and cowpea extracts by western blotting after SDS PAGE and native isoelectric focusing (IEF). The subunit molecular mass of these proteins ranged from 14–23 kDa in leaf, root and stem tissues, but higher molecular mass forms were observed in germinated seed (cotyledon) extracts. After IEF, multiple forms of stathmin-like proteins were observed in different tissues from all plant extracts. In general, the pIs ranged from 3.5 to 5.0, although each tissue showed a unique IEF pattern of stathmin-like proteins. Stathmin-like proteins in plants appear to be similar to mammalian stathmin in size, pI, and immunological determinants.

Moraxella catarrhalis: clinical significance, antimicrobial susceptibility and BRO beta-lactamases.

Moraxella catarrhalis is an important pathogen of humans. It is a common cause of respiratory infections, particularly otitis media in children and lower respiratory tract infections in the elderly. Colonisation of the upper respiratory tract appears to be associated with infection in many cases, although this association is not well understood. Nosocomial transmission is being increasingly documented and the emergence of this organism as a cause of bacteremia is of concern. The widespread production of a beta-lactamase enzyme renders Moraxella catarrhalis resistant to the penicillins. Cephalosporins and beta-lactamase inhibitor combinations are effective for treatment of beta-lactamase producers, and the organism remains nearly universally susceptible to the macrolides, fluoroquinolones, tetracyclines and the combination of trimethoprim and sulfamethoxazole. Two major beta-lactamase forms, BRO-1 and BRO-2, have been described on the basis of their isoelectric focusing patterns. The BRO-1 enzyme is found in the majority of beta-lactamase-producing isolates and confers a higher level of resistance to strains than BRO-2. The BRO enzymes are membrane associated and their production appears to be mediated by chromosomal determinants which are transmissible by an unknown mechanism. The origin of these novel proteins is unknown.

The high alkaline fraction on isoelectric focusing of cerebrospinal fluid is cystatin C

A high alkaline fraction with a p I of 9.2 is sometimes seen on isoelectric focusing patterns of cerebrospinal fluid. The appearance of this fraction mainly depends on the type of concentrators used to prepare the cerebrospinal fluid samples, prior to isoelectric focusing. The amino acid sequence of the high alkaline fraction showed sequence identity to cystatin C, a cysteine protease inhibitor with a p I of 9.2–9.3 and a molecular mass of 13.4 kDa. In addition, on Western blot the high alkaline fraction was recognized by an antibody, directed against cystatin C. Taken together, the present findings demonstrate that the high alkaline fraction is cystatin C.

Degradation of myocardiac myosin and creatine kinase in rats given alkaline ionized water.

Recently, the authors have shown that marked necrosis and fibrosis of myocardium were observed in rats given alkaline ionized water (AKW). To clarify the cause of myocardial lesions, the activities of myosin ATPase, actomyosin ATPase and creatine kinase (CK) in myocardium of rats given AKW at 15 weeks-old were compared with those in myocardium of rats given tap water (TPW). Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of myocardiac myosin and isoelectric focusing (IEF) of myocardiac CK were performed which revealed a distinct difference between AKW and TPW groups. The activities of myosin ATPase and actomyosin ATPase in the AKW group were higher than those in the TPW group, and these elevated activities were caused by the degradation of myosin in the AKW group judging from the SDS-PAGE pattern of myosin. On the other hand, the activity of CK in the AKW group was lower than that in the TPW group, and the IEF pattern of CK showed leakage of myocardiac CK. These results indicate that increases in actomyosin ATPase activity and myosin ATPase activity, plus the decrease in CK activity caused the disorder of coupled reaction in male rats given AKW at 15 weeks-old. It is concluded that this disorder of coupled reaction may cause marked myocardiac necrosis and fibrosis in rats given AKW.

Amino acid and protein changes in tilapia and spanish mackerel after irradiation and storage

Some amino acids in tilapia decreased while some others increased when subjected to doses up to 10.0 kGy. However, 10 kGy contributed to a significant reduction in all amino acids of Spanish mackerel. Variations in amino acid contents continued during post-irradiation storage with no consistant trend of increase or decrease. SDS-PAGE of protein from both fish showed 27 bands of subunits with MW < 14.0–94.0 KD. Isoelectric focusing patterns of sarcoplasmic protein of unirradiated and irradiated fish showed no charge in the number of bands, while some changes were observed in the intensities of the anodic and cathodic bands depending on isoelectric points (pIs).

Variability of bothrojaracin isoforms and other venom principles in individual jararaca ( Bothrops jararaca) snakes maintained under seasonally invariant conditions

Bothrojaracin (BJC) is a potent thrombin inhibitor isolated from the venom of Bothrops jararaca. Venoms from individual snakes have been shown to vary in BJC content, and more than one molecular variant (isoform) has been identified in the same venom. In order to determine whether the production of this protein and its isoforms varies under seasonally invariant conditions, an analysis was made of BJC isolated from venoms collected individually once a month for 10 months from two female B. jararaca snakes kept under conditions of constant temperature and photoperiod. The crude venom from each individual snake exhibited a characteristic pattern of protein bands on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), with no noticeable variation throughout the collecting period. BJC from individual venoms was purified by gel filtration on Sephacryl S-200 followed by an affinity column (PPACK-thrombin Sepharose). BJC content and other activities such as phospholipase A 2, azocaseinolytic activity and inhibition of thrombin-induced platelet aggregation varied considerably among the samples. Purified BJC from both snakes inhibited fibrin coagulation and migrated as a single band of 27,000 mol. wt on SDS–PAGE. However, the BJC pattern on non-denaturing PAGE differed between the two snakes, with four to six bands per sample each month, which were all recognized by polyclonal anti-BJC antibodies. The isoelectric focusing pattern of BJC was also characteristic for each snake, with only minor differences throughout the collecting period. These results indicate that under seasonally invariant conditions: (1) there was a considerable variation over the 10-month period in the production of BJC and other important venom activities such as phospholipase A 2 and proteinases; (2) individual B. jararaca snakes produced a distinctive array of BJC isoforms; and (3) despite quantitative differences, there were essentially no qualitative differences in the production of BJC isoforms by individual snakes during the 10-month period.

Rapid purification and properties of human glycodelin (endometrial α 2-globulin)

The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at M r 28 000 after sodium dodecyl sulphate–polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with p I values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of β-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of α-helix conformation.

Mitochondrial ALDH Polymorphism Affects Ethanol-Derived Acetate Disposition in Wistar Rats

We studied the relationship between polymorphism of aldehyde dehydrogenase (ALDH) and ethanol and acetate kinetics in male Wistar rats. Blood ethanol and acetate concentration time curves after intravenous bolus administration of 1 g/kg of ethanol were estimated by moment analysis. ALDH isozymes in hepatic subcellular fractions were detected by isoelectric focusing (IEF)- Blood acetate profiles were divided into two patterns: one-peak type and two-peak type. IEF studies on ALDH isozymes in subcellular fractions showed polymorphism of cytosolic and mitochondrial ALDHs. Polymorphism patterns of ALDH2 corresponded with patterns of blood acetate profiles. Significant differences in mean residence time of acetate and the ratio of area under curves (AUCs) of acetate to ethanol (AUCacetate/AUCEtoH) occurred between different IEF patterns of mitochondrial low Km ALDH (ALDH2). Therefore, it was clarified that ALDH2 polymorphism affects ethanol-derived acetate disposition in Wistar rats. Similarity of AUC values of acetate considered, these observations suggest that ALDH2 polymorphism results in change of effects of acetate and acetate-generated adenosine on the central nervous system and other organs during chronic ethanol consumption.

Mitochondrial ALDH Polymorphism Affects Ethanol‐Derived Acetate Disposition in Wistar Rats

We studied the relationship between polymorphism of aldehyde dehydrogenase (ALDH) and ethanol and acetate kinetics in male Wistar rats. Blood ethanol and acetate concentration time curves after intravenous bolus administration of 1 g/kg of ethanol were estimated by moment analysis. ALDH isozymes in hepatic subcellular fractions were detected by isoelectric focusing (IEF). Blood acetate profiles were divided into two patterns: one-peak type and two-peak type. IEF studies on ALDH isozymes in subcellular fractions showed polymorphism of cytosolic and mitochondrial ALDHs. Polymorphism patterns of ALDH2 corresponded with patterns of blood acetate profiles. Significant differences in mean residence time of acetate and the ratio of area under curves (AUCs) of acetate to ethanol (AUCacetate/AUCEtOH) occurred between different IEF patterns of mitochondrial low K(m) ALDH (ALDH2). Therefore, it was clarified that ALDH2 polymorphism affects ethanol-derived acetate disposition in Wistar rats. Similarity of AUC values of acetate considered, these observations suggest that ALDH2 polymorphism results in change of effects of acetate and acetate-generated adenosine on the central nervous system and other organs during chronic ethanol consumption.

Alpha-2-HS-glycoprotein (AHSG) polymorphism in semen and saliva.

Polymorphism of alpha-2-HS-glycoprotein (AHSG) was demonstrated in human semen and whole saliva samples by thin-layer polyacrylamide gel isoelectric focusing (IEF) and immunoblotting. Although the seminal AHSG IEF patterns were found to differ from those of plasma AHSG from the corresponding donors, incorporation of Nonidet P-40 into the IEF gel (pH 4.2-4.9) enabled us to phenotype seminal AHSG correctly. Salivary AHSG, however, exhibited IEF patterns similar to those of the corresponding plasma AHSG. By treating the samples with neuraminidase, it was possible to determine the AHSG types using 2-5 microL semen and 50-100 microL whole saliva samples. The AHSG types determined separately in 47 sets of semen, whole saliva, urine and plasma samples from the same donors correlated perfectly with each other. AHSG typing could, therefore, provide an additional discriminant characteristic in the forensic examination of semen and saliva samples.

Qualitative assessment of intrathecal IgG synthesis by isoelectric focusing and immunodetection: Interlaboratory reproducibility and interobserver agreement

Detection of intrathecal IgG synthesis is important in patients with suspected multiple sclerosis (MS). The recommended method for the detection of intrathecal synthesis of IgG is isoelectric focusing and immunodetection of oligoclonal bands. Recently "The Committee for European Concerted Action for Multiple Sclerosis" has recommended that the results of isoelectric focusing for the detection of intrathecal synthesis of oligoclonal bands should not only be stated as positive or negative for intrathecal synthesis; instead, the laboratory should provide a detailed description of the IgG pattern in both cerebrospinal fluid (CSF) and serum together with a conclusion concerning the presence of intrathecal synthesis. We studied the interlaboratory reproducibility and interobserver agreement of isoelectric focusing, and the recommended classification system for the assessment of intrathecal IgG synthesis, in two separate patient groups employing kappa statistics. We found a high degree of interlaboratory reproducibility (133 patients; kappa = 0.95 +/- 0.05) and interobserver agreement (356 patients; kappa = 0.97 +/- 0.04) when the presence or absence of intrathecal IgG synthesis was assessed. The agreement was less pronounced, although still fully satisfactory, when the results were classified according to the detailed system by two laboratories (133 patients; kappa = 0.86 +/- 0.08) and two observers (356 patients; kappa = 0.88 +/- 0.08). Two specific problems in the interpretation of isoelectric focusing patterns were identified: one related to the discrimination of a pattern with several closely spaced bands which may represent a monoclonal protein; the other related to determining whether systemic band synthesis was present. We conclude that isoelectric focusing and immunodetection is a very reproducible technique for the detection of intrathecal IgG synthesis. Well defined criteria and extensive standardization may, however, be necessary when more elaborate classification systems are employed.