Nutan T. Patel

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. K m,app values for MPTP and benzylamine, a preferred MAO B substrate, were 316 and 64 μM, respectively. 4‐Phenyl‐1,2,3,6‐tetrahydropyridine (PTP), the nor derivative of MPTP, was also a substrate (K m,app = 221μM). MPDP+ MPTP, and MPP+, but not PTP, were found tobe 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.

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.

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.

RNA associated with nonhistone chromosomal proteins of dog liver

Abstract The nonhistone chromosomal proteins were separated on Sephadex G-200 into 3 fractions of which two were associated with 3S RNA. The RNA eluted with fraction I (guanine + cytosine content 54%) is tightly bound to the proteins from which it can be separated only after digestion with pronase. The RNA associated with fraction III (guanine + cytosine content 64%) can be separated from the proteins directly by chromatography on DEAE-Sephadex A 25. No dihydropyrimidines have been detected in any of the two RNAs.

Protein composition of liver nuclear ribonucleoprotein particles of rats fed carcinogenic aminoazo dyes

Abstract The feeding of carcinogenic 3′-methyl-4-dimethylaminoazobenzene to rats alters the protein composition of liver nuclear 30S ribonucleoprotein particles which are proposed to be involved in the processing and transport of the newly synthesized RNA. After 10 weeks of feeding of the carcinogenic aminoazo dye, one of the major proteins is missing from these particles but not from the particles isolated from liver of animals fed with noncarcinogenic 4-aminoazobenzene. In all the groups of rats studied, the RNA associated with the isolated particles was of high specific activity.

Purification of human platelet monoamine oxidase B by high performance liquid chromatography

Abstract Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chromatography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0–1%) of octyl-β-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently labeled with [3H]-pargyline [3H(G)] or catalytic activity using [14C-methylene]-benzylamine as substrate. [3H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit Mr (approximately 59,000) of MAO B were...

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).

Characterization of low molecular weight nonhistone chromosomal protein from dog liver

The nonhistone chromosomal proteins (NHC-proteins) are viewed as the possible positive effecters of the transcription control in eukaryotic cells [ 1 ] . The reproducible isolation and characterization of individual NHCproteins are essential in order to explore their function in the control of gene activity. In this communication we describe the isolation and characterization of an electrophoretically homogeneous low molecular weight NHC-protein from dog liver.