Studies on catechol-O-methyltransferase in rat brain using two-dimensional gel electrophoresis

The presence of catechol-O-methyltransferase (COMT) in the rat brain was studied using a combination of two-dimensional gel electrophoresis (2-DE), protein blotting and a specific antiserum. Two major immunoreactive proteins were identified—one with mol. wt 23 kdalton and an isoelectric point of 5.2, the other of mol. wt 25 kdalton and an isoelectric point of 5.1. In addition, multiple lower molecular weight immunoreactive proteins, possibly corresponding to breakdown products of the enzyme, were also detected. The 23 kdalton form of COMT, which is probably the soluble form of the enzyme, is a major protein visible on silver-stained 2-D gels of rat brain. In contrast, the other proteins recognized by the antiserum were not detected by the silver stain. These results demonstrate, using 2-DE, that at least two distinct forms of catechol-O-methyltransferase are present in rat brain. In addition, since one of these proteins is stained by silver, these results also serve to identify another protein visible on 2-D electrophoretograms of rat brain.     

Isolation of the mRNA encoding rat liver catechol-O-methyltransferase

A highly specific, well characterized rabbit antiserum to purified rat liver catechol-O-methyltransferase (COMT; EC 2.1.1.6) and the procedure of polysome immunoadsorption have been used to isolate a messenger RNA which encodes a single polypeptide when translated in vitro. Western blotting and immune fixation have shown multiple active forms of the enzyme to exist; a major, soluble one with MW of 23,000 and pI of 5.2 and another, membrane-bound one with MW of 26,000 and a pI of 6.2 (1). When translated in vitro, the purified message synthesizes a protein of molecular weight (MW) 23,000 and pI 5.2, values in agreement with those for purified enzyme reported by other investigators (2,3). Only the soluble form is seen after in vitro translation; the other immunoreactive proteins possibly arise due to post-translational modifications which do not occur in the lysate; or perhaps another mRNA exists. Cloning of the COMT cDNA will resolve this issue and should be feasible in light of our data indicating that the mRNA isolated here represents 0.46% of total rat liver polyadenylated message.     

Heterogeneity of renin substrate released from hepatocytes and in brain extracts

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17β estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000–65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.     

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.     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.     

Prothrombin biosynthesis: characterization of processing events in rat liver microsomes

Plasma and hepatic microsomal forms of rat prothrombin have been compared by sodium dodecyl sulfate-polyacrylamide electrophoresis and isoelectric focusing. The major prothrombin species that accumulated in the microsomes of rats treated with warfarin had a molecular weight of 78 500 and a pI in 8 M urea of 6.3-6.5. Plasma prothrombin had a molecular weight of 83 500 and a pI of 5.3-5.7. Microsomes from normal rat liver contain a second pool of precursor with a molecular weight of 83 500, and digestion with the glycosidase Endo H indicated that this form has been processed to contain complex carbohydrates, while the Mr 78 500 form is a high mannose form and is the substrate for the vitamin K dependent carboxylase. Treatment of rats with tunicamycin revealed that glycosylation was not essential for carboxylation or secretion from the liver. Comparison of the aglyco forms of prothrombin and its precursors suggests that the intracellular forms contain a basic, Mr approximately 1500 peptide that is missing from the plasma form of prothrombin.     

Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BPrCl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BPrCl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BPrCl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH7777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is highly conserved.     

Investigation of proteases with chromogenic substrates after isoelectric focusing (IEF)

Summary A technique is described for the detection of protease isoenzymes which is more sensitive than disc electrophoresis. Supernatants of crude rat and human organ homogenates are subjected to analytical isoelectric focusing (IEF) and the gel strips are finally incubated in histochemical media containing 4-methoxy-2-naphthylamine amino acids or peptides and diazonium salts for simultaneous or post-coupling. The incubation media are identical with those used for section histochemistry of proteases. This combination of IEF and proteases histochemistry yields excellent and reproducible data which cannot be obtained by protease histochemistry alone. Post-coupling delivers less and more diffuse bands than simultaneous coupling. For simultaneous coupling, Fast Blue B and Fast Black K are the most suitable diazonium salts. More bands are found in agarose gels compared with polyacrylamide. Sex-differences exist for endopeptidases in the submandibular gland, but are absent in other rat organs. Despite their uniform membrane localization in tissue sections, aminopeptidase (AP) A and M and dipeptidylpeptidase (DPP) IV and -glutamyltranspeptidase (GGT) show striking heterogenous band patterns depending on the investigated organ. The similar band patterns of APA and APM can be specified by the use of activators or inhibitors. In rat kidney, up to 26 bands are obtained with DPP II and IV substrates, 3 for APA and APM and up to 12 for GGT. DPP IV of human liver is different from that in rat liver.     

Two-dimensional polyacrylamide gel electrophoresis analysis of phosphorylated, membrane-localized ras p21 proteins

The ras p21 oncogene product migrates as a heterogeneous series of polypeptides as resolved by both one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). We have prepared polyclonal rat serum antibody to ras p21 and used this as well as monoclonal antibodies to immunoprecipitate forms of p21 synthesized in vivo in transformed NIH3T3 cells. Two-dimensional PAGE of p21 resolved two distinct groups of polypeptides, one acidic (pI 4.8-5.3) which we call the "A" forms, and one less acidic or more basic (pI 6.5-7.0) which we call the "B" forms. It is the membrane-localized, B forms of v-ras-Ha p21 that are predominantly phosphorylated in vivo.     

Isoelectric analysis of 3H-pargyline-labelled monoamine oxidase in rat and carp

1. After selective binding of [3H]pargyline to either monoamine oxidase (MAO) A or MAO B in the rat liver, MAO B alone in the rat brain and MAO in carp brain and liver, molecular weight and isoelectric points (pI) of these MAO were determined by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing and results obtained were compared. 2. For all tissues tested, SDS-polyacrylamide gel electrophoresis of [3H]pargyline-bound samples revealed a labelled protein band of an apparent mol. wt of 60,000 da. 3. Estimation of radioactivity of [3H]pargyline bound after isoelectric focusing revealed a single protein band with acidic pI values of about 5.5 for rat brain and liver MAO B. 4. Moreover, the pI values of about 7.5 were obtained for carp brain and liver MAO. This basic value was also found for MAO A in the rat liver MAO A.     

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.     

Cross-linking study on localization of the binding site for elongation factor 1 alpha on rat liver ribosomes

Complexes containing rat liver 80 S ribosomes, poly(uridylic acid), phenylalanyl-tRNA, elongation factor 1 alpha, and guanylyl(beta, gamma-methylene)-diphosphonate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 26 fractions by chromatography on carboxymethylcellulose. Each protein fraction was subjected to diagonal polyacrylamide-sodium dodecyl sulfate gel electrophoresis. Four cross-linked pairs containing elongation factor 1 alpha were on the vertical line below the diagonal. The ribosomal protein spot of each pair was cut out from the gel plate and labeled with 125I. The labeled proteins were extracted from the gel and identified by two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both 60 S and 40 S subunits were identified: L12, L23, L39, S23/S24, and S26, three proteins of which had been found to be cross-linked also to elongation factor 2 (Uchiumi, T., Kikuchi, M., Terao, K., Iwasaki, K., and Ogata, K. (1986) Eur. J. Biochem. 156, 37-44). These results afford direct evidence that both elongation factors interact with partially overlapping sites on rat liver ribosomes.     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

Isoelectric focusing and 2D electrophoresis of the human androgen receptor.

Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polyacrylamide gels before and after photoaffinity labeling with [3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 +/- 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated in IEF sample buffer containing no salt, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 +/- 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with Mw 90-95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with Mw 90-95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc+) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and Mw 90-95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc+) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Isoelectrofocusing Electrophoretic Analysis on Soluble Protein of Germinated Embryos and Young Shoots from Different Cytoplasmic Male Sterile Lines and Their Maintainers in Wheat

The soluble proteins of germinated embryos and young shoots in Wheat were studied by means of isoelectrofocusing (IEF) electrophoresis. The materials used were three species of cytoplasmic male-sterile (CMS) lines (Type E, T and A) and their maintainers. The protein quantity of pI 4. 9 in male-fertile materials is higher than the corresponding male-sterile ones; the protein of pI 6.85 is probably the result of gene expression in Timopheevi cytoplasm; the protein of pI 7.6 is a typical band of Jinfeng male-sterile line. Evident differences on soluble protein chromatograms of IEF electrophoresis were observed both in germinated embryos and young shoots which were from different kinds of CMS line, these differences could be used as indexes identifying various CMS lines.     

Heat-shock proteins and nucleolar hypertrophy in the liver of rat infused with methionine-free total parenteral nutrition

Infusing a methionine-free solution into rats for 7 days resulted in a marked enlargement of liver nucleoli. By the analysis of two-dimensional gel electrophoresis, a spot 'a' (76 kDa, pI 5.3) stained with Coomassie blue was observed to accumulate highly in liver cytosol from rat infused with methionine-free solution. Metabolically labeling experiments with [35S]methionine showed that 'a' was more heavily labeled in primary hepatocytes of rats infused with methionine-free solution than in those of control rat. To ascertain whether 'a' is one of stress proteins, primary hepatocyte cultures were incubated at 42 degrees C for 2 h. 'a' (76 kDa, pI 5.3) was slightly induced in control hepatocytes but not appreciably in hepatocytes from the treated rat. In contrast, two other spots 'b' (74 kDa, pI 5.6) and 'c' (74 kDa, pI 5.3) were highly induced at 42 degrees C in hepatocytes from control and treated rats. The antibody against the consensus sequence peptide of hsp70 family reacted with 'a' (76 kDa, pI 5.3) as well as 'b' and 'c'. Immunoblot analysis revealed that 'a' accumulates highly in hepatocytes of treated rats. These results indicate that infusion of methionine-free solution into rats induces one member of the hsp70 family in hepatocytes.     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

粗毛栓菌胞外锰过氧化物酶的纯化及性质

培养于麦草粉上的白腐担子菌粗毛栓菌分泌胞外木质纤维素降解酶(纤维素酶、木聚糖酶、漆酶、锰过氧化物酶和木质素过氧化物酶)。经过超滤、盐析、离子交换层析、凝胶过滤和活性聚丙烯酰胺凝胶电泳等步骤,获得了初步纯化的锰过氧化物酶组分。利用变性聚丙烯酰胺凝胶电泳和等电点聚焦技术所测定的锰过氧化物酶的相对分子质量和等电点分别为35.7 ku和pI 2.8。研究结果表明,所纯化的锰过氧化物酶在407nm处具有最大光吸收峰,该酶最适作用pH值和温度分别为pH 5.3和35℃。