Molecular Weight Determination of Component Polypeptides of Glutenin after Fractionation by Gel Filtration

Reduced and cyanoethylated glutenin was fractionated into three fractions (F I, F II and F III) by gel filtration on Sephadex G–100 in 0.1 m acetic acid. The molecular weight determination was made with these three fractions by sedimentation equilibrium in 6.5 m guanidine hydrochloride containing 0.01 m acetic acid. The molecular weight obtained was 44,000 for F II, and 32,000 for F III. F I showed a distribution of molecular weight due to the aggregation. The average molecular weight of F I was 52,000, being 27,000 at the meniscus and 98,000 at the bottom. The estimation of molecular weight by SDS–PAGE* gave overestimated values for glutenin polypeptides, as was already reported for gliadin.     

Composition of a Photosystem I chlorophyll protein complex from Anabaena flos-aquae

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Characterization of the filamentous hemagglutin from Bordetella pertussis by gel electrophoresis

Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S₂ (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S₂, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.     

Polyacrylamide Gel Electrophoresis of Plasteins

A peptic hydrolysate of soybean protein was filtered with Sephadex G–25 and was separated approximately into four fractions (I, II, II, and IV in the order of mol. wt.). Fraction II (av. mol. wt: 1043) and III (av. mol. Wt.: 685) were more plastein-productive than others. When plastein produced from Fraction II with Nagarse was investigated by plate electrophoresis using 7.5% polyacrylamide-gel, the upper limit of the molecular weight was found to be about 25,000. A similar result was obtained also with Fraction III. The increase of molecular weight in the course of the plastein formation with the mixture (substrate) of Fractions II and III was shown that the final product lay mainly in a position between cytochrome c (mol. wt.: 11,700) and Nagarse (mol wt.: 27,600). In addition, the gel-electrophoretic experiments revealed that the most favorable condition for the plastein synthesis were pH 6.5 and 35% in substrate concentration.     

Composition of a photosystem I chlorophyll protein complex from Anabaena flos-aquae.

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Fractionation of Some Bovine Whey Proteins by Recycling Gel Filtration on a Large Scale

Fractionation of bovine whey concentrate was performed by gel filtration on Sephadex G-75 both on a laboratory scale and on a large scale. By a recycling procedure an improved separation was obtained and the whey proteins were resolved into four fractions in the weight ratio 3:12:1:4.

The fractions were analysed by polyacrylamide gel (PAG) electrophoresis and the apparent molecular weights were determined by thin layer gel chromatography (TLG) and by sodium dode-cylsulfate (SDS) gel electrophoresis.     

东亚飞蝗主要过敏原的分析、鉴定与纯化

通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离东亚飞蝗Locusta migratoria manilensis(Meyen)的蛋白质组分并测定其分子量,收集过敏病人血清,采用免疫印迹(Western—blotting)法鉴定其过敏原成分,通过凝胶过滤层析对东亚飞蝗过敏原进行分离纯化。结果表明:东亚飞蝗蛋白粗提液条带大概有30条左右,其中主带大约有10条,相对分子量约为13、15、25、28、40、45、55、70、100、110ku,其中蛋白含量最丰富的约在70ku左右。免疫印迹结果显示,蝗虫过敏条带主要有5条,相对分子量分别约为19、29、38、70、130ku。通过凝胶过滤层析对东亚飞蝗过敏原进行分离纯化,得到了一个高纯度相对分子质量约为70ku东亚飞蝗过敏原,并且发现了一个相对分子质量约为130ku的蝗虫新过敏原。本研究为临床上蝗虫食物变态反应性疾病的诊断和治疗奠定基础。     

Plasmodium berghei: I. Immunochemical properties of fractions of a soluble extract

Soluble extracts of Plasmodium berghei were separated into 12 fractions following preparative disc electrophoresis in polyacrylamide gel. One or two protein bands were detected in each fraction by analytical disc electrophoresis. Similarly, one or two precipitinogens were generally detected in each of Fractions 1 through 11 by immunoelectrophoresis and by double immunodiffusion in agar gel, while the unfractionated extract contained 10 precipitinogens. Antisera produced in rabbits against each fraction each contained two or three (sometimes five) antiplasmodial precipitins demonstrable by immunoelectrophoresis. Serial fractions obtained in separate runs were closely similar to each other, although some degree of overlapping sometimes occurred between neighboring fractions. Glycoproteins were detected in all the fractions, but chiefly in Fractions 4 and 12. The bulk of the RNA in the extract was located in Fraction 4, while hemoglobin was usually confined to Fraction 6. The molecular weights of the soluble components of P. berghei range between 8000 and 130,000.     

Isolation of 3-Hydroxy-β-ionol from Burley Tobacco

Foxtail millet (Setaria italica) proteins were fractionated into five fractions, i. e., water-, salt-, ethanol-, sodium dodecyl sulfate (SDS)- and 2-mercaptoethanol (ME)-soluble fractions, by successive extraction with various solvents from millet flour. The proportion in each fraction was 7.2, 5.6, 40, 25 and 20% respectively, of total flour nitrogen. The proteins of the ethanol- and SDS-soluble proteins were similar in amino acid composition and molecular weight distribution. More than 15 different molecular weight classes of proteins ranging from 11,000 to 150,000 were distinguished by SDS-polyacrylamide gel electrophoresis without prior reduction of their disulfide bonds. These major protein bands in the gel were estimated to be homo-olygomers (monomer, dimer, trimer, etc.) of subunit A or subunit B. The molecular weights of subunits A and B were 12,000 and 17,000, respectively. Subunits A and B were also different in amino acid composition: subunit A had higher content of methionine.     

Types of Amylases in Rice Grains

The subunit structure of phenoloxidase from the larvae of housefly, Musca domestica vicina Maquart, was investigated by urea treatment, SDS-polyacrylamide gel electrophoresis and electron microscopy. Phenoloxidase was dissociated into a single type of subunit (MW 6.5 × 10⁴) by 5 m urea treatment. A similar subunit (MW 6.1 × 10⁴) was also detected by SDS-polyacrylamide gel electrophoresis. Furthermore, four additional protein bands with molecular weights of 4.8 × 10⁴, 3.5 × 10⁴, 2.4 × 10⁴ and 1.2 × 10⁴ were detected by SDS-polyacrylamide gel electrophoresis by extensive incubation of phenoloxidase with SDS. A cylindrical molecular structure was deduced from the electron microscopic analysis. The outside and inside diameter and the height of phenoloxidase molecule were evaluated to be 100, 50 and 70 Å, respectively.     

Purification and Subunit Structure of DNA-dependent RNA Polymerase III from Wheat Germ

A rapid and simple, large-scale method for the purification of DNA-dependent RNA polymerase III (EC 2.7.7.6) from wheat germ is presented. The method involves enzyme extraction at low ionic strength, polyethyleneimine fractionation, (NH₄)₂SO₄ precipitation, and chromatography on DEAE-Sepharose CL-6B, DEAE-cellulose, and heparin agarose. Milligram quantities of highly purified enzyme can be obtained from kilogram quantities of starting material in 2 to 3 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that RNA polymerase III contains 14 subunits with molecular weights of: 150,000; 130,000; 94,000; 55,000; 38,000; 30,000; 28,000; 25,000; 24,500; 20,500; 20,000; 19,500; 17,800; and 17,000. Subunit structure comparison of wheat germ RNA polymerases I, II, and III indicates that all three enzymes may contain common subunits with molecular weights 20,000, 17,800, and 17,000. In addition, RNA polymerases II and III may contain a common subunit with a molecular weight of 25,000, and RNA polymerases I and III may contain a common subunit with a molecular weight of 38,000.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

PURIFICATION OF BOVINE PINEAL HYDROXYINDOLE O-methylTRANSFERASE BY IMMUNOADSORPTION CHROMATOGRAPHY

A procedure is described for the use of immunoadsorption chromatography of hydroxyindole O-methyltransferase (HIOMT). HIOMT was purified from bovine pineal extract by affinity chromatography on immunoglobulins (Ig)-Sepharose. The overall purification was about 45-fold; the yield was 84%. This enzyme constitutes about 2.0% of the soluble proteins in the pineal gland. The enzyme represented a single precipitin line on Ouchterlony double diffusion plate and immunoelectrophoresis. Ultracentrifugation analysis indicated the existence of molecular aggregates of enzyme and disc gel electrophoresis showed one main protein band and several minor bands. However sodium dodecyl sulphate (SDS) gel electrophoresis showed a single protein band with subunit molecular weight 38,000 demonstrating bovine pineal HIOMT to be polymer enzyme of a single subunit. The properties of the purified enzyme including disc gel electrophoretic pattern, the effect of pH, chemicals and substrates and immunological properties were identical with those of the crude enzyme.     

Comparative analysis of the lipopolysaccharides of a rough and a smooth strain of Pseudomonas syringae pv. phaseolicola

The lipopolysaccharides (LPS) of a rough (R) and a smooth (S) strain of Pseudomonas syringae pv. phaseolicola were analysed. The S-LPS revealed markedly more rhamnose and fucose, but less glucose, than the R-LPS. The presence of 3-O-methyl-rhamnose (acofriose) in the S-LPS was confirmed by cochromatography with authentic acofriose. SDS polyacrylamide gel electrophoresis of the S-LPS demonstrated a cluster of regularly spaced high molecular weight fractions, which was almost lacking in the R-LPS. The main fatty acids of the lipid A of both LPS species were 3-OH-10:0,3-OH-12:0,2-OH-12:0, and 12:0. Two N-linked diesters were demonstrated: 3-O(12:0)-12:0 and 3-O(2-OH-12:0)-12:0. S-LPS was subjected to mild hydrolysis and the degraded polysaccharide separated into three fractions by gel permeation chromatography on a Fractogel TSK HW-50 column. Fraction I, representing nearly only the O-specific side chain, consisted of rhamnose and fucose in a molar ratio of 4:1, with 4% of the rhamnose being 3-O-methylated (acofriose). Fraction II, representing mostly core material, was composed of glucose, rhamnose, heptose, glucosamine, galactosamine, alanine, and a still unidentified amino compound, in an approximate molar ratio of 3:1:1:1:1:1:1, and KDO. Fraction III consisted of released monomers and salts. The LPS was highly phosphorylated (3.28% phosphorus in the core fraction). The thus characterized composition of the LPS O-chain seems to be unique for the pathovar phaseolicola of P. syringae, although many similarities exist to other pathovars as well as to other bacterial species.Abbreviations LPS lipopolysacchairdes - GC/MS combined gas liquid chromatography-mass spectrometry - HVE high voltage electrophoresis - KDO 2-keto-3-deoxyoctonic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate P.s. pv. phaseolicola is termed P. phaseolicola in the text     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Isolation and substrate specificities of five chitinases from the hepatopancreas of Northern shrimp, Pandalus borealis

Five enzymes designated chitinase I, IIa, IIb, III, and IV have been isolated from the hepatopancreas of Pandalus borealis in a procedure including column chromatography on Q-Sepharose, Sephacryl S-200, phenyl-Superose and Superdex 75. The isolated enzymes were analysed by SDS PAGE. Chitinase I, III, and IV gave only one major band corresponding to 54–55 kDA. Chitinase IIa showed one major band at 61 kDA and two diminutive bands at 17 and 55 kDa, while chitinase IIb gave two major bands at 17 and 44 kDa. Estimated by gel filtration, the native molecular weights of chitinase I, IIa, IIb, III, and IV were 61, 69, 39, 57, and 54 kDa, respectively. The substrate and reaction specificities of the isolated chitinases were investigated, and the results show that the isolated enzymes are true chitinases. They do not hydrolyse N,N′-diacetylchitobiose or p-Nitrophenyl-N-acetyl-β-D-glucosaminide, but express activities when longer chitooligosaccharides or nitrophenylated chitooligosaccharides are used as substrates. Chitinase I and IIa gave an initial random cleavage pattern and might be classified as endochitinases, while chitinase III and IV released dimeric units from the substrates and might be termed chitobiosidases.     

Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle

A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.     

Copper-binding proteins and copper tolerance in Pisam sativum L.

The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 M Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 M Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of M_r 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of M_r 3700, and a copper-protein (III-2) with an M_r, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly-isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (M_r 3700), III-2 (M_r 66000), and IV (M_r 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.Abbreviations DEAE diethylaminoethyl - M_r relative molecular mass - SDS sodium dodecyl sulfate - PAGE polyacrylamide-gel electrophoresis J.M. Palma was the recipient of a research fellowship from the Caja General de Ahorros y Monte de Piedad de Granada and CSIC. We are grateful to Dr. J. Moreno-Carretero, R + D Department, UNIASA, Granada, for conducting the amino-acid analyses. This work was supported by grant 603/275 from CAICYT-CSIC (Spain).     

Pig liver monoamine oxidase: Studies on the subunit structure

The molecular weight of pig liver MAO has previously been shown to be about 115,000 with 1 mole of covalently bound FAD per mole of enzyme. Gel filtration of purified enzyme on Sepharose 4B in 6 m guanidine and 0.1 m mercaptoethanol (MCE) and analytical ultracentrifugation in 0.1% sodium dodecyl sulfate (SDS) and 0.1% MCE yielded molecular weights of 55,000 and 63,000, respectively. By polyacrylamide electrophoresis in 0.1% SDS + MCE one band of 60,000 MW appeared. These results seem to imply that the enzyme is composed of two subunits of which one carries the active site. If MCE was omitted during the gel electrophoresis two equally large bands of about 60,000 MW were formed. By using enzyme inhibited by [¹⁴C]pargyline, a MAO-inhibitor blocking the active site of the enzyme in a 1:1 molar ratio, it was found, however, that both bands contained pargyline. Furthermore, amino acid analyses yielded the same amino acid composition of the two bands. The results are interpreted that the enzyme is composed of two subunits of identical molecular size (about 60,000) of which only one contains the active site and that the enzyme preparation contained two forms of the enzyme presumably differing in the number of disulfide bonds.