Identical Subunits Of Molecular Mass Research Articles

Purification and properties of methanol dehydrogenase and aldehyde dehydrogenase from Methylobacillus glycogenes.

Methanol dehydrogenase (MDH) and aldehyde dehydrogenase (ALDH) were purified to a homogenous state from Methylobacillus glycogenes, an obligate methylotroph. MDH (Mr 140, 000) was composed of two different subunits (Mr 60, 000 and 9, 000) forming an α2/β2 structure. MDH was indicated as a metalloquinoprotein containing one atom of calcium (Ca) per enzyme molecule. Binding of Ca was so tight that it was hard to remove Ca completely without denaturation of enzyme protein. A partially resolved enzyme resumed its original enzyme activity upon exogenous addition of Ca. Purified ALDH (Mr 144, 000) was composed of two identical subunits of molecular mass of 72, 000. ALDH was proved to be a quinoprotein in which PQQ is bound covalently.

Purification and characterisation of an inducible ?-galactosidase from Corynebacterium murisepticum

The beta-galactosidase (EC 3.2.1.32) of Corynebacterium murisepticum (inducible by lactose and galactose) was purified by successive column chromatography on Sephadex G-200, DEAE-Sephadex A-50 and DEAE-cellulose (DE52). The enzyme was found to be a dimer of identical subunits of molecular mass 100,000 daltons. The Km values of the enzyme for the substrates lactose and o-nitrophenyl-beta-D-galactopyranoside (ONPG) are 16.7 mM and 4.4 mM, respectively, indicating, its low affinity for the substrates. The Ouchterlony immunodiffusion method exhibited immunological homogeneity of the enzyme preparation. The catalytic site of the enzyme does not take part in antigen-antibody reaction.

An NADP/thioredoxin system in leaves: Purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach

An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells. Until now there was no evidence for this system in green leaves. Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species. Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves. This represents the first time that NTR has been characterized from a plant source. Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein ( M r 68,000) composed of two subunits of identical molecular mass ( M r 33,000) that resembled Escherichia coli NTR immunologically. Spinach thioredoxin h existed in two forms ( M r, of 13,500 and 12,000) and was highly specific for plant NTR. Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves. Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties.

Purification and characterization of a serine hydroxymethyltransferase from an obligate methylotroph, Hyphomicrobium methylovorum GM2.

A serine hydroxymethyltransferase was purified to complete homogeneity from a serine-producing obligate methylotroph, Hyphomicrobium methylovorum GM2. The enzyme has a molecular mass of about 98 kDa and consists of two subunits of identical molecular mass. The holoenzyme exhibits absorption maxima at 280 nm, 340 nm and 415 nm in potassium phosphate buffer, pH 7.3, the last of which shifts with a change in pH (6.0-7.5) and contains 2 mol pyridoxal phosphate/mol enzyme. The holoenzyme is converted to the apoenzyme on incubation with phenylhydrazine and reconstituted on the addition of pyridoxal phosphate. The enzyme activity was inhibited on the addition of several sulfhydryl-modifying reagents and then recovered with 2-mercaptoethanol. One sulfhydryl group per subunit was found to be responsible for the activity. Isoelectric focusing showed that the enzyme has a pI of 5.6. The Km values for glycine, L-serine and DL-beta-phenylserine are 0.046 mM, 0.15 mM and 33 mM respectively.

Purification and characterization of rat liver glutaminase

Phosphate-dependent glutaminase (EC 3.5.1.2) from livers of starved rats was purified about 400-fold to near homogeneity. The specific activity of the final pool was more than 30 U/mg protein. For the rapid quantification of the enzyme activity a simple and sensitive assay, based on the determination of the produced ammonia with an o-phthalaldehyde reagent, was developed which avoids massive dilution of the samples. The enzyme preparation involved extraction of the enzyme from sonified isolated mitochondria after treatment with a brief hypotonic shock followed by ammonium sulphate precipitation, ion-exchange and hydroxyapatite chromatography. A major improvement was the stabilization of the enzyme by chymostatin protecting it from degradation by a protease of presumably lysosomal origin. In the presence of chymostatin or leupeptin the half-life of glutaminase in a crude mitochondrial preparation subsequent to mild treatment with digitonin could be increased to more than 200 h. The relative molecular mass of the protein (Mr 170,500) was estimated by sucrose gradient ultracentrifugation. The molecular mass of the subunits (Mr 57,000) was determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These results suggest a protein composed of three subunits of identical molecular mass. The molecular data clearly differentiate liver glutaminase from the phosphate-dependent glutaminase present in kidney.

Purification and properties of a nonheme bromoperoxidase from Streptomyces aureofaciens.

The first bacterial nonheme type bromoperoxidase has been purified to homogeneity from the chlorotetracycline-producing actinomycete Streptomyces aureofaciens Tü 24. Purification was accomplished by (NH4)2SO4 precipitation, DEAE-cellulose chromatography at different pH-values, and molecular sieve chromatography. The purified enzyme has a molecular mass of 90 to 95 kDa based on ultracentrifugation and gel filtration. The enzyme is composed of three subunits of identical molecular mass (m = 31 kDa). Bromoperoxidase catalyses the bromination of monochlorodimedone, but not its chlorination, and has no peroxidase or catalase activity. The optimum pH is 4.5. The enzyme does not exhibit an absorption peak in the Soret region of the optical spectrum. X-ray fluorescence spectroscopy revealed that the enzyme does not contain any metals in equimolar amounts. Bromoperoxidase is stable in a pH range from pH 4.0 to pH 10.0 at 4 degrees C for weeks and does not loose any activity when incubated at 80 degrees C for 2 h.

Purification and characterization of camel liver glutathione S-transferase

1. 1. Glutathione S-transferases have been purified (18-fold) in 65–70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose. 2. 2. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5. 3. 3. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes. 4. 4. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

Rat liver nuclear protein kinases NI and NII. Purification, subunit composition, substrate specificity, possible levels of regulation.

Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein-phosvitin-Sepharose column step, which separates the enzymes from the other chromosomal non-histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non-histone proteins with partly different specificities as shown by two-dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides. Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity. Several chromosomal proteins coeluted with NII from the casein-phosvitin-Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein-NII complex was incubated with ATP.