scholarly journals The purification and properties of the soluble cytochromes c of the obligate methylotroph Methylophilus methylotrophus

1980 ◽  
Vol 192 (2) ◽  
pp. 421-427 ◽  
Author(s):  
A R Cross ◽  
C Anthony
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Low Molecular Weight ◽  
Redox Potentials ◽  
Obligate Methylotroph ◽  
Methylophilus Methylotrophus ◽  
Midpoint Potential ◽  
Purification And Properties ◽  
Cytochromes C ◽  
Cytochrome Cl

The obligate methylotroph Methylophilus methylotrophus contains three distinct soluble cytochromes c. The major cytochromes, cytochrome cH (about 50% of the total) and cytochrome cL (about 42%), were similar in most respects to the cytochromes cH and cL of the facultative methylotroph Pseudomonas AM1 [O'Keeffe & Anthony (1980) Biochem. J. 192, 411-419]. Cytochrome cH had a high isoelectric point, a midpoint redox potential at pH 7.0 of 373 mV and a low molecular weight (8500). The cytochrome cL had a low isoelectric point, a midpoint potential of 310 mV and a molecular weight of 21,000. The third cytochrome, cytochrome cLM, was clearly distinct from cytochromes cH and cL. Like the cytochrome cL of Pseudomonas AM1, the cytochrome cL of M. methylotrophus had the lowest midpoint potential, it reacted most rapidly with methanol dehydrogenase and it combined to the greatest extent with CO. Cytochromes cH and L of M. methylotrophus differed from those from Pseudomonas AM1 in having unusually high midpoint redox potentials for non-photosynthetic bacteria and in exhibiting a split alpha-band at low temperatures.

scholarly journals The two cytochromes c in the facultative methylotroph Pseudomonas AM1

1980 ◽  
Vol 192 (2) ◽  
pp. 411-419 ◽  
Author(s):  
David T. O'Keeffe ◽  
Christopher Anthony
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Isoelectric Point ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Redox Potentials ◽  
Midpoint Potential ◽  
Cytochromes C

It was previously suggested that there is only one soluble cytochrome c in Pseudomonas AM1, having a molecular weight of 20000, a redox midpoint potential of about +260mV and a low isoelectric pint [Anthony (1975) Biochem. J.146, 289–298; Widdowson & Anthony (1975) Biochem. J.152, 349–356]. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 has now revealed the presence of two different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isoelectric focusing. Molecular weights were determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behaviour of the two cytochromes on ion-exchange celluloses. The more abundant cytochrome cH (λmax. 550.5nm) had a low molecular weight (11000), a midpoint potential of about +294mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The less abundant cytochrome cL (λmax. 549nm) was about 30% of the total; it had a high molecular weight (20900), a midpoint potential of about +256mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The pH-dependence of the midpoint redox potentials of the two cytochromes c were very similar. There were four ionizations affecting the redox potentials in the pH range studied (pH4.0–9.5), two in the oxidized form (pK values about 3.5 and 5.5) and two in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the two propionate side chains of the haem. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C1 compounds, although still able to grow well on multicarbon compounds such as succinate. Whether or not these two cytochromes c have separate physiological functions is not yet certain.

scholarly journals The electron-transport chains of the obligate methylotroph Methylophilus methylotrophus

1980 ◽  
Vol 192 (2) ◽  
pp. 429-439 ◽  
Author(s):  
Andrew R. Cross ◽  
Christopher Anthony
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Redox Potentials ◽  
Obligate Methylotroph ◽  
Methylophilus Methylotrophus ◽  
Cytochrome O ◽  
Cytochromes C ◽  
Band Absorption ◽  
Membrane Bound ◽  
Α Band

The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O2-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J.192, 421–427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with α-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b563 in methanol-limited bacteria. A third b-type cytochrome with an α-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a3, which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O2 in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

scholarly journals Purification and Properties of Staphylocoagulase

1975 ◽  
Author(s):  
A.D. Muller ◽  
B. M. Bas ◽  
H. C. Hemker
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Aspartic Acid ◽  
Acid Composition ◽  
Isoelectric Point ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino

Staphylocoagulase, an exoprotein of coagulase positive staphylocoagulase, has been purified to a state in which only trace amounts of contaminating proteins are detectable.Purification was more than 35,000 fold, which is 7 times more than the highest value reported in the literature. The yield was about 15%.Aspartic acid was found as a single N-terminal amino acid in this preparation. The molecular weight is 61,000 and the isoelectric point lies at pH 4.53.The amino acid composition was determined.

scholarly journals Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight

1975 ◽  
Vol 151 (2) ◽  
pp. 227-238 ◽  
Author(s):  
A G McLennan ◽  
H M Keir
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Euglena Gracilis ◽  
Deoxyribonucleic Acid ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Optimum Conditions ◽  
Electrophoretic Mobilities ◽  
Substrate Activation ◽  
Purification And Properties

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an ‘activated’-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5′-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 × 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

Purification and properties of human low molecular weight kininogen

1982 ◽  
Vol 706 (2) ◽  
pp. 145-152 ◽  
Author(s):  
Werner Müller-Esterl ◽  
Magdalene Vohle-Timmermann ◽  
Barbara Boos ◽  
Brigitte Dittman
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight ◽  
Purification And Properties

Purification and Properties of Rat α2 Acute-Phase Macroglobulin (α2M)

1979 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
J. J. Emeis ◽  
J. Hemmink
Keyword(s):  
Molecular Weight ◽  
Acute Phase ◽  
Deae Cellulose ◽  
Rat Plasma ◽  
Low Molecular Weight ◽  
Purification Procedure ◽  
Purification And Properties ◽  
Purification Scheme ◽  
Pure Amino Acid ◽  
Proteolytic Activities

For our studies on the relation between blood fibrinolytic activity and repair of mechanically damaged arteries in our rat model we need a specific and sensitive assay for α2M. in the rat α2M is an acute-phase protein of which the level in blood is normally near zero but increases as a result of the damage. Moreover α2M is known to inhibit proteases involved in the fibrinolytic system. We developed a new purification procedure in which, conditions known to be harmful to the functionality of α2M were avoided. α2M was purified from plasma of turpentine-treated rats and proteolytic activities were suppressed throughout the purification procedure. The purification scheme successively involves: rivanol precipitation, Con A-Sepharose chromatography and DEAE-cellulose chromatography. Thus 48 mg of α2M was obtained from 100 ml rat plasma i.e. 20% recovery. The preparations were biochemically and immunologically pure. Amino acid and carbohydrate compositions were determined. The molecular weight is 760.000. The molecule consists of 4 subunits, M.W. = 190.000. A 1%1cm = 8.8 and p1 = 4.8. It binds 1 mole of trypsin or plasmin per mole. Bound proteases were only active on low molecular weight substrates such as BAEE and BOC-L-val-gly-L-arg βNA. Kinetic data of the bound enzymes (pH-optimas, Km and Vmax) indicate that factors other than steric hindrance are involved in the inhibitory action of α2M.

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

1990 ◽  
Vol 69 (2) ◽  
pp. 129-131 ◽  
Author(s):  
Kaoru Takegawa ◽  
Satoshi Miki ◽  
Takayuki Jikibara ◽  
Shojiro Iwahara
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight ◽  
Purification And Properties

scholarly journals The sialic acid content and isoelectric point of human kininogen

1975 ◽  
Vol 145 (2) ◽  
pp. 401-403 ◽  
Author(s):  
J C Londesborough ◽  
U Hamberg
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Amino Acid ◽  
Isoelectric Point ◽  
Acid Content ◽  
Low Molecular Weight ◽  
Sialic Acid Content

The sialic acid content of highly purified human kininogen was found to be about 8.6 mol/mol(mol.wt. 50,000). The isoelectric point (pH 4.9 +/- 0.2) is much higher than that of bovine low-molecular-weight kininogen, but is close to that expected from the amino acid and sialic acid analyses.

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