Human liver MAO-A and MAO-B separated by immunoaffinity chromatography with MAO-B-specific monoclonal antibody.

A monoclonal antibody was used to prepare immunoaffinity columns that efficiently bind monoamine oxidase B activity but not monoamine oxidase A activity from detergent extracts of human liver mitochondria. The only discrete polypeptide component that eluted from affinity columns with potassium thiocyanate migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 59,000, as expected for human monoamine oxidase B. These results support the hypothesis that there is an intrinsic structural difference between monoamine oxidase A and B and demonstrate that immunoaffinity chromatography can physically resolve the two enzyme species in liver extracts.

Dependence of the composition of the protein moiety of nuclear ribonucleoprotein particles on the extent of particle purification as studied by electrophoresis including a two-dimensional procedure.

Extraction with 0.1 M NaCl in 0.01 M Tris-HCl buffer, pH 8.0 releases from liver nuclei 30-40-S ribonucleoprotein particles containing newly synthesized RNA. Separation of the protein moiety of these particles by acid-urea gel electrophoresis depends on the concentration of beta-mercaptoethanol in the buffer used for the solubilization of the particles. At low concentration or with short time of solubilization, only a polypeptide chain with apparent molecular weight 38 000 penetrates into the gel and can be detected by electrophoresis. By introduction of two-dimensional polyacrylamine gel electrophoresis, we succeeded to separate the protein moiety of these particles into a core group of 4 major and 6 minor polypeptides with molecular weights ranging from 38 000 to 50 000 and a second group of 19 polypeptides ranging in molecular weight from 50 000 to 120 000. The composition of the protein moiety of these particles is dependent on the extent of purification. Polypeptides with molecular weight below 50 000 represent 55% of the total protein of particles purified only by centrifugation through a 15-30% sucrose gradient. If the particles were first purified by gel filtration through Bio-Gel A-50m followed by centrifugation in sucrose gradient, the low molecular weight proteins represent 80% of all the proteins of the particles. The purification removed selectively the minor high molecular weight polypeptides without resulting in any extensive release of the four major polypeptides with molecular weight below 50 000 which form a stable core particle. By repeated purification it is possible to strip the particles of the high molecular weight polypeptides even further. An increase in the NaCl concentration of the extraction buffer to 0.35 M will extract additional 30-40-S particles associated with a newly synthesized RNA from the cell nucleus. These particles contain the same polypeptides as particles extracted at lower salt concentration. Extraction with 0.1 M and 0.35 M NaCl at pH 8.0 removed from the nucleus approximately 55% of all RNA labeled in 30 min after intraperitoneal injection of [3H] orotic acid to the rats.

Polysomal poly(A)+ RNA in liver of rats fed 3'-methyl-4-dimethylaminoazobenzene and in hepatoma induced by the same carcinogen.

Feeding of carcinogenic azo dyes to rats results in a release into the cytoplasm of RNA sequences which in liver cells of control animals are degraded in the cell nucleus. A cross-hybridization of polysomal poly(A)+ RNA from liver of rats fed the hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene and from liver of control animals with their complementary DNA has shown, that the disruption of the processing and/or release of nuclear RNA induced by the carcinogen is not reflected in a change in the polysomal poly(A)+ RNA. After 17 weeks of feeding the hepatocarcinogen, there is no difference in the sequence complexity of polysomal poly(A)+ RNA in the liver. It is therefore not probable that the RNA sequences released from the nucleus by the azo dye serve as a template for protein synthesis. An alteration in the polysomal poly(A)+ RNA population was observed only temporarily at an earlier stage of feeding of the azocarcinogen. It coincided with the regeneration of the liver in a response to the initial toxic effect of the azocarcinogen. Therefore, it is probable that this alteration is the result of a temporary change in the population of liver cells. A cross-hybridization of liver and hepatoma complementary DNA with the polysomal poly(A)+ RNA from both organs have shown an overlap of the polysomal poly(A)+ RNA sequences of the hepatoma with the sequences present in the liver, with many liver sequences missing, or present only in very low concentration in the hepatoma.

Isolation and characterization of the predominant protein in nuclear ribonucleoprotein particles from rat liver.

The predominant protein of the nuclear ribonucleoprotein particles of rat liver was isolated by polyacrylamide gel electrophoresis. The polypeptide represented 35% to 40% of the total mass of the protein moiety. Its molecular weight was estimated to be 38 000 and its NH2-terminal residue was found to be threonine. The amino acid composition is unique in having a high content of glycyl residues (20%) and NG-dimethylarginine (14% of total arginyl residues).

Heat shock protein is tightly associated with the recombinant human glucocorticoid receptor:glucocorticoid response element complex.

The association of heat shock proteins (hsp) with steroid hormone receptors may have functional significance for steroid receptor action. The association of hsp90 with steroid receptors is thought to maintain the receptors in the nonactivated state until their interaction with the respective ligand. The association of hsp70 with progesterone receptor has been well documented. However, there is evidence both for and against the association of hsp70 with the glucocorticoid receptor (GR). We have examined the interaction between hsp70 and human (h) GR over-expressed in the Baculovirus system. Immunoprecipitation and sucrose gradient centrifugation studies demonstrated the association of hsp70 with both the nonactivated and in vitro activated hGR. We were unable to dissociate hGR and hsp70 by incubation of crude cytosol with 3 mM ATP and 0.5 M NaCl. In vivo activation of hGR did not result in dissociation of hsp70 from hGR. Hsp70 coeluted with hGR from a glucocorticoid response element (GRE)-Sepharose column, suggesting that hsp70 is part of the GRE-hGR complex. Both an anti-hGR antibody and an anti-hsp70 antibody were capable of further retarding the migration of a [32P]GRE-hGR complex in polyacrylamide gels. The in vitro activated hGR has been shown to be highly active in an in vitro transcription system. We speculate that hsp70 may influence the DNA-binding and/or transcriptional activities of hGR.

Human oxysterol-binding protein. I. Identification and characterization in liver.

The function of cellular oxysterol-binding proteins is not known. Because of the proposed receptor-like role of these proteins in regulating cholesterol synthesis and the importance of the liver in the cholesterol metabolism of the body, we have studied the oxysterol-binding sites found in specimens of human liver. A protein that binds with high affinity to certain oxygenated derivatives of cholesterol has been identified and characterized in several ways. When 25-hydroxy-[3H]cholesterol is used as ligand, specific binding components can be identified in cytosolic liver extracts fractionated on sucrose density gradients in the fractions sedimenting at 4S and 7S. Cholesterol and steroid hormones do not compete for these binding components, but several oxysterols do. In cytosolic extracts, all binding appears to be a single class kinetically, with an apparent Kd of 28 x 10(-9) mol/L. We found the protein responsible to have a pI of about 4.8. Purification of the protein allowed preparation of an antiserum and subsequent immunoprecipitation of a photoaffinity-labeled component from crude cytosolic extracts. Electrophoresis of the immunoprecipitates revealed a single specifically labeled protein band, with a mol wt of about 57,000. The protein described here may have a regulatory role in the synthesis of cholesterol in the human.

Metabolism of the neurotoxin in MPTP by human liver monoamine oxidase B.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was oxidized to dihydropyridine MPDP+ and pyridine MPP+ by preparations of monoamine oxidase B (MAO B), including pure human liver MAO B:monoclonal antibody complex, Km,app values for MPTP and benzylamine, a preferred MAO B substrate, were 316 and 64 microM, respectively. 4-Phenyl-1,2,3,6-tetrahydropyridine (PTP), the nor derivative of MPTP, was also a substrate (Km,app = 221 microM). MPDP+, MPTP, and MPP+, but not PTP, were found to be irreversible inhibitors of MAO B. Our studies support the hypothesis that MPTP is oxidized in primate brain by MAO B to MPDP+, which is then converted to MPP+, a major metabolite found in the substantia nigra.

Azocarcinogen-induced release of chromatin-associated RNA into liver cytoplasm: the possible role of reiterated RNA sequences in the control of RNA processing.

In the liver of rats fed the azocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'MeDAB) reiterated RNA sequence transcribed from middle repetitive DNA are released into the cytoplasm. The same repetitive nucleotide sequences can be isolated from the chromatin of the liver of control animals in the form of metabolically highly active, 13 000 daltons RNA. This small, chromatin-associated RNA originates from nuclear RNA larger than 10 S. The discontinuation of the feeding of the azocarcinogen will not stop the release of the nuclear reiterated RNA sequences into the cytoplasm. The repetitive sequences of nuclear RNA which are released into the cytoplasm in animals fed the azocarcinogen can no longer be found in the chromatin in the form of small RNA molecules. The results can be explained by the assumption that the reiterated RNA sequences are involved in the upholding of RNA processing. A cell-specific processing of RNA will be maintained by the interaction of reiterated RNA fragments from already processed RNA with the reiterated complementary sequences on RNA yet to be processed. Existence of such a feed-back circuit would make it possible to explain how a temporary interference of the azocarcinogen with RNA processing will result in the disappearance of specific reiterated RNA sequences from the chromatin. It could also explain the continuation of the release of the same repeated RNA sequences into the cytoplasm as part of larger RNA molecules even after the removal of the carcinogen.

Protein composition of nuclear ribonucleoprotein particles isolated from liver of rats in the early stages of feeding of 3'-methyl-4-dimethylaminoazobenzene and from hepatoma induced by the same carcinogen.

The feeding of carcinogenic 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) in the early stages results in a change in the protein composition of the nuclear ribonucleoprotein particles of the rat liver. These particles are associated with newly synthesized RNA and it is assumed that they are involved in the processing and in the transport of this RNA. After 6 weeks of feeding of this azocarcinogen, the amount of one of the main polypeptides (apparent molecular weight 42 000) is decreased and after 10 weeks of feeding the particles are devoid of this polypeptide completely. Feeding of the non-carcinogenic p-aminoazobenzene (AB) is without any effect. The loss of this polypeptide is not characteristic for the malignant transformation. In the nuclear ribonucleoprotein particles isolated from hepatoma which has been induced by 3'-MeDAB this polypeptide is present in even higher proportion to other polypeptides than it is in particles isolated from liver cells of control animals. The 3'-MeDAB binds to the proteins of the liver nuclear ribonucleoprotein particles and interferes with the RNA processing. It is proposed that the changes in the composition of the protein moiety of the particles reflect changes in the population of liver cells leading finally to the selection of hepatoma cells which are resistant to the toxic effect of 3'-MeDAB on RNA processing.

RNA associated with non-histone chromosomal proteins in rat liver and in ascites hepatoma.

The purpose of the experiments was to determine if changes in the post-transcriptional processing of RNA in the hepatoma also affect the low molecular weight nuclear RNA which is transcribed from families of related genes and associated with non-histone chromosomal proteins. Separation of non-histone chromosomal proteins on Sephadex G-200 into three fractions separated the low molecular weight RNA associated with these proteins into metabolically stable RNA firmly bound to the first eluted fractions of high molecular weight non-histone chromosomal proteins, and into a metabolically active RNA which is eluted with the third protein fraction and can be separated from the low molecular weight non-histone chromosomal proteins by chromatography on DEAE-Sephadex A 25 (fraction III RNA). In liver as well as in hepatoma, this fraction III RNA represents about 50% of the RNA associated with the non-histone chromosomal proteins. Fraction III RNA from both tissues has an approximate molecular weight of 13,000, is rich in guanylic acid, is lacking dihydropyrimidines and is copied from the repetitive sequences of DNA. The content of uridylic acid is much higher in fraction III RNA isolated from hepatoma than in the same RNA isolated from liver, and competitive hybridization has shown that hepatoma fraction III RNA contains not only new base sequences which are not present in liver fraction III RNA, but also lacks some sequences which are present in the liver RNA. The technique of RNA/DNA hybridization in the presence of competing RNA has shown that, in hepatoma, the cytoplasmic RNA competes with more than 60% of the fraction III RNA for the hybridization sites on repetitive DNA. No competition was found when liver cytoplasmic RNA was used. The low ratio of competing hepatoma cytoplasmic RNA or of liver or hepatoma nuclear RNA which is required to displace fraction III RNA from its hybridization with DNA indicates that this RNA is synthesized and in hepatoma is also released into the cytoplasm as a part of larger RNA molecules. The detection of the nucleotide sequences found in liver associated with non-histone chromosomal proteins in the cytoplasm of hepatoma cells is evidence for extensive disruption of the post-transcriptional control in hepatoma.

Isolation of pure, catalytically active human liver monoamine oxidase B: antibody complex.

Monoamine oxidase B was purified from human liver mitochondria using a monoclonal antibody, MAO B-1C2, which recognizes monoamine oxidase B but not A. Triton X-100 extracts of mitochondria were incubated with purified MAO B-1C2 (IgG1), and the catalytically active enzyme:antibody complex was isolated by affinity chromatography on Protein A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex revealed the presence of four polypeptide bands (monoamine oxidase B, 57,900 dalton; antibody heavy chain, 52,200 dalton; and two light chains, 29,400 and 27,700 dalton), and indicated a 1:1 stoichiometric ratio of enzyme to antibody. This method gave 154-fold purification of the enzyme from mitochondria.

Characterization of the human oxysterol receptor overexpressed in the baculovirus system.

Oxysterols are potent regulators of enzymes of the de novo cholesterol biosynthetic pathway and do not require the LDL (low density lipoprotein):LDL receptor system for their regulatory actions. The search for an alternate transduction system led to the identification of an oxysterol binding protein. This cytosolic protein has been extensively characterized, purified, and cloned. Although it fulfills the pharmacologic criteria for an oxysterol receptor by binding to oxysterols with affinities corresponding to their regulatory potencies, its function in maintaining cholesterol homeostasis has not been determined. We have overexpressed the human oxysterol receptor in Spodoptera frugiperda cells using the Baculovirus system. The overexpressed protein binds oxysterols, but not cholesterol. The affinity for 25-hydroxycholesterol determined by competitive binding assay was 7.3 +/- 4.4 nM (mean +/- SD), and the relative affinities of several other oxysterols approximately corresponded to their potencies in cell systems. The expressed protein migrated as a single immunoreactive band on denaturing polyacrylamide gels with a molecular mass of 94 kDa. The molecular mass calculated from sucrose gradient centrifugation and gel filtration was 273 kDa for the 9.8S form, 217 kDa for the 7.8S form, and 184 kDa for the 6.6S form. However, velocity gradient centrifugation and heparin-sepharose chromatography each indicated that there were at least two fractions containing specific oxysterol binding. We conclude that we have successfully overexpressed the human oxysterol receptor and that biochemical analysis of the overexpressed protein provides evidence of interactions with other proteins. Further analysis of the overexpressed protein should provide clues regarding its role in maintaining cholesterol homeostasis.

A monoclonal antibody elicited to human platelet monoamine oxidase. Isolation and specificity for human monoamine oxidase B but not A.

We have isolated a mouse monoclonal antibody to human platelet monoamine oxidase (MAO) B. The antibody (MAO-1C2) was isolated from a fusion of mouse myeloma P3/X63 Ag8 to spleen cells from a BALB/c mouse immunized with a partially purified platelet preparation in which an estimated 21-31% of the protein was [3H]pargyline-labeled MAO B. The antibody indirectly immunoprecipitates both [3H]pargyline-labeled, catalytically inactive human MAO B, and unlabeled, catalytically active human MAO B. Binding of the antibody to MAO B has no detectable effect on catalytic activity. MAO-1C2 is specific for human MAO B, and fails to immunoprecipitate MAO A indirectly from human placenta or liver. Its ability to immunoprecipitate human MAO B but not MAO A from extracts of human liver provides a convenient technique for separating the two forms of the enzyme for comparative studies. The antibody does not recognize mouse liver MAO B, suggesting that the determinant is not universally expressed on MAO B from all species.

Use of a monoclonal antibody for comparative studies of monoamine oxidase B in mitochondrial extracts of human brain and peripheral tissues.

Monoamine oxidase B (MAO B; EC 1.4.3.4) activity in detergent extracts of mitochondria from autopsy brain (gray matter and medulla), liver, lung, and kidney from a single individual and from pooled, human platelets could be immunoprecipitated by a monoclonal anti-human platelet MAO B antibody (MAO-1C2) in combination with appropriate secondary reagents. MAO A activity, which was detected in brain, liver, lung, and kidney, was not immunoprecipitated under the same conditions. All MAO B-containing extracts, regardless of tissue source, inhibited immunoprecipitation of [3H]pargyline-labeled human platelet MAO, and the shapes of the inhibition curves were identical. The concentration of immunologically detectable MAO B protein in the extracts was estimated from immunoprecipitation competition data by reference to a standard curve relating observed inhibition of immunoprecipitation to the concentration of catalytically active platelet MAO added (estimated from [3H]pargyline binding data). MAO B protein concentrations measured by this radioimmunoassay were similar to concentrations of active MAO B as measured by pargyline binding. These results demonstrate that in the brain and peripheral tissues studied, molecules with MAO B activity share a unique antigenic determinant and similar catalytic efficiency. They also extend previous observations that MAO B molecules extracted from mitochondria bear an antigenic determinant which is not present on MAO A molecules. These results demonstrate the validity of a new competitive radioimmunoassay for active plus inactive MAO B concentration in human platelet extracts and extracts of mitochondria from human tissues. This radioimmunoassay should complement [3H]pargyline binding assays and enzyme activity assays in studies designed to clarify the mechanisms of genetic, disease, and treatment factors which lead to differences in MAO B function among individuals.

Isolation, characterization, and application of monoclonal antibodies to rat tyrosine hydroxylase.

Tyrosine hydroxylase (TH, tyrosine 3-monooxygenase; EC 1.14.16.2) activity in crude extracts of rat pheochromocytoma, rat brain, and bovine adrenal medulla can be immunoprecipitated in an indirect assay by monoclonal antibodies prepared against partially purified rat pheochromocytoma TH. One of these monoclonal antibodies, TH-2D8-2, can be used for immunocytochemical localization of TH in cell bodies, dendrites, and axons in catecholaminergic neurons (e.g., cells in the substantia nigra) of rat brain and in the cell body, neurites, and growth cones of rat pheochromocytoma cells after treatment with nerve growth factor. When linked to Affi-gel 10, this monoclonal antibody can also be used for immunoaffinity purification of rat and bovine TH. These results suggest that TH-2D8-2 is a valuable reagent with which to investigate the localization, physiological regulation, and function of this important enzyme.