Molecular weight determination by gel filtration

MPO from H. niger was isolated and its molecular weight determined by gel filtration (135 kDa) and the SDS-PAGE analysis showed that the enzyme is a dimmer [140]. MPO has also been obtained from other plant speeies such as D. stramonium [141] and N. tabacum [142], Inhibition studies of Hyoscyamus MPO demonstrated the quinoprotein characteristic of this enzyme [140], a feature previously reported for N. tabacum MPO, after conducting a suicidal inhibition of the enzyme with phenyUiydrazine [143]. 

A deoxyadenosylcobalamin-dependent ribonucleoside triphosphate reductase has been partially purified from cell free extracts of the extreme thermophile, Thermus X-l 14). The enzyme preparation catalyzed the reduction of GTP and CTP at comparable rates, while UTP and ATP were reduced at only one-tenth the rate of GTP reduction. Only the dithiols could serve as reducing substrates. The enzyme has a temperature optimum of 70°, and the allosteric regulation of the enzyme activity is also temperature-dependent. The reduction of ATP is specifically stimulated by dGTP only at a higher temperature. Maximum stimulation of ATP reduction is observed at approximately 75°, while no stimulation can be detected at 37°. The molecular weight determined by gel filtration was approximately 80,000 but no information about the subunit structure is yet available. 

A j8-D-2-acetamido-2-deoxyglucosidase from E. coli has been purified to near homogeneity by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate and urea. Studies of the substrate specificity of the purified enzyme (pH optimum 7.7, Km (for 4-nitrophenyl 2-acetamido-2-deoxy-l3-D-gluco-pyranoside 0.43 mmol 1 ) confirmed that it has an exo-action. The molecular weight (determined by gel filtration and by gel electrophoresis) of the enzyme in a dissociating medium is 2.6 x 10, showing that it does not contain subunits. 

Figure 3. Molecular weight determination using gel filtration HPLC. Protein standards (see methods) are represented by ( ) and enzyme activity by (O). Enzyme activity corresponds to a molecular weight of approximately 70,000 daltons.

In the original method, the separation between the free ligand and the complex is based on the difference of size. When there is simultaneous fixation of two ligands of similar molecular weights, separation by gel filtration is difficult or impossible. The respective concentrations of each ligand can be determined if they have different optical characteristics or if one of them is radioactive. In the particular (but frequent) case of an ADP-ATP mixture, the situation is unfavourable same optical spectra, similar sizes. In order to avoid important levels of radioactivity, we have adapted the Hummel and Dreyer method by using anion exchange separation of the protein and of the two nucleotides. 

The molecular weight determined by the gel filtration chromatography with Toyopearl 55 HW was 115 kdal while that estimated by SDS-PAGE was 43 kdal (Figure 4,5). 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

The sedimentation coefficient is 3.0 S. The approximate molecular weight of the enzyme was determined by gel filtration (117) to be about... 

In 1972 Ogawa and Toyama (56) purified three components— A-I-a, A-I-b, and A-II-1—which were adsorbed on a gauze column during purification from Cellulase Onozuka P1500, a commercial preparation of T. viride cellulase. These three components had molecular weights of 32,000, 48,000, and 48,000 as determined by gel filtration and contained 7-16% carbohydrate. Each is reported to carry out the random hydrolysis of CM-cellulose and to degrade hydrocellulose (Avicel) and cellooligosaccharides except for cellobiose. The order of reactivity toward either cotton or Avicel was A-II-1 > A-I-b > A-I-a. The proteins adsorbed on cellulose comprised 38% of the total cellulase protein. 

Tomato PG I and PG II are similar in many respects. Both enzymes are endo-PG s although PG II is more effective in reducing the viscosity of pectate (30). Their pH optima are near 4.5, and both enzymes exhibit broad peaks of activity extending from pH 1.5 to 5.5 when hydrolyzing short-chained substrates (30). They are basic glycoproteins with pH s of 8.6 and 9.4 for PG I and PG II, respectively (12). Antibodies raised against PG II react also with PG I (32). The same polypeptide is obtained when PG I and PG II are denatured in SDS solutions (12., 36). However, the enzymes differ markedly in molecular size and stability to heat. The molecular weights, as determined by gel filtrations are 100,000 and... 

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

Four isolectins have been isolated from bulbs of Crocus sativus with approximately molecular weight 48 KDa as determined by gel filtration chromatography [42]. Rivoflavine and thiamin are also constituents of saffron [43]. Very recently, anthocyanins were identified in the flowers of Crocus sativus [44],... 

In contrast the enzyme purified from lupin root nodule cytosol is reported to consist of a single polypeptide chain of molecular weight 235,000 (Boland and Benny, 1977). No information is available at present regarding the possible subunit structure of other eukaryote glutamate synthases although the ferredoxin-dependent enzyme from V. faba has a molecular weight of 150,000, as determined by gel filtration (Wallsgrove et al., 1977) and the pyridine nucleotide-dependent enzyme of Saccharomyces cerevisiae has a sedimentation coefficient of 14.6 S (Roon et al., 1974). 

Analytical techniques The apparent molecular weight of the isolated polysaccharide fraction was determined by gel filtration (HPLC). Dextrans were used as standards. 

The molecular weight of cathepsin B from various organs and tissues is in the range of 24,000-28,000. The highest values in this range have been determined by gel filtration methods. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme is often nicked between 23,000 and 5000 and showed multiple forms on electrofocusing (p/ 4.5-5.6). The N-terminal amino acid of rat liver cathepsin B is leucine. It contains carbohydrate 19) but the exact sugar content has not yet been measured. 

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