Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection

Iersel, Jack van; Jzn, J. Frank; Duine, Johannis A.

doi:10.1016/0003-2697(85)90072-7

Cited by 81 publications

(27 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…On the other hand, UV absorption spectroscopy gave A2S0nm = 13.5, as calculated with a formula published by 1% van Iersel et al [18]. Using the molecular mass of 220 kDa, the calculated molar extinction coefficient was e=300000 M -1 .cm -1 at 280 nm.…”

Section: Oqpi:iga Stoichiometry and ~ Of The Complexsupporting

confidence: 70%

Inhibition of neutrophil elastase by the α₁‐proteinase inhibitor‐immunoglobulin A complex

Adam

Bieth

1996

FEBS Letters

View full text Add to dashboard Cite

Neutrophil elastase is thought to be involved in cartilage destruction occurring in rheumatoid arthritis despite the local presence of al-proteinase inhibitor. Part of synovial fluid al-proteinase inhibitor forms a mixed disulfide with immunoglobulin A, which has been postulated to lack inhibitory activity. We show here that the immunoglobulin-inhihitor complex tightly inhibits neutrophil elastase and catbepsin G, bovine pancreatic trypsin and chymotrypsin, and porcine pancreatic elastase. Although the rate constant of inhibition of neutrophil elastase by immunoglobulin A-bound al-proteinase inhibitor (kass = 9.2 × 10 5 M -l" s -I) is about 10-fold lower than that measured with the free inhibitor, it is high enough to enable efficient inhibition of elastase in vivo.

show abstract

Section: Oqpi:iga Stoichiometry and ~ Of The Complexsupporting

confidence: 70%

Inhibition of neutrophil elastase by the α₁‐proteinase inhibitor‐immunoglobulin A complex

Adam

Bieth

1996

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…(1 mg ml-l) value close to that of wild-type MDH (2.03 vs 2.04) (van Iersel et al, 1985). In contrast, gel-filtration of the purified native protein under denaturing conditions showed that it does not contain a P-subunit.…”

Section: Purijication and Subunit Structure Of Mdh And Related Proteimentioning

confidence: 64%

Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Methylophaga marina

Janvier

Frank

Luttik

et al. 1992

Journal of General Microbiology

View full text Add to dashboard Cite

Methanol oxidation in Mefhyfophuga marina appears to be organized in a way similar to that in other Gramnegative methylotrophs, involving a quinoprotein methanol dehydrogenase (MDH; EC 1.1.99.8) and two soluble cytochromes. The MDH is composed of two different subunits, most probably arranged in an a2P2 structure. The two cytochromes are of the e-type and differ in size (molecular mass 19.5 and 12.5 kDa) and isoelectric point (PI 4.6 and 9.2). The one with the lowest isoelectric point, commonly designated as cytochrome eL, is able to oxidize reduced MDH. Taking advantage of the ability of M. marina, a restricted facultative methylotroph, to utilize fructose as a growth substrate, mutants impaired in methanol utilization were isolated after application of optimal concentrations of ethylmethane sulphonate. Three classes of methanol oxidation mutants were obtained. Class I mutants were affected in a global regulation of the synthesis of both apo-methanol-dehydrogenase and cytochrome cL as well as PQQ (pyrroloquinoline quinone). Class I1 mutants did not produce an active MDH, but instead a comparable amount of a 65 kDa protein was found in the cell-free extract upon SDS-PAGE. This mutant protein was purified and compared to wild-type MDH. It was located in the periplasm, but unlike MDH it was composed of only two identical large subunits. Each of these subunits was able to bind one molecule of PQQ. An antiserum raised against wild-type MDH did not react with the mutant protein. Conversely, an antiserum raised against mutant protein weakly cross-reacted with wild-type MDH, suggesting that the presence of the P-subunit in MDH dramatically changes its immunochemical behaviour. Three of the class I1 mutants did not produce PQQ. In the presence of PQQ, partial revertants able to grow on methanol medium were obtained (class 111). Class 111 mutants produced a stable apo-MDH consisting of a-and P-subunits and showing a normal reaction with the anti-MDH serum. Although this apo-MDH can bind PQQ, enzymic activity was not restored in nitro. This suggests that, in addition to apo-MDH and PQQ, other factors are required for the assembly of an enzymically active MDH.

show abstract

“…The specific absorption coefficients of MDH and activator protein at 280 nm were determined from the absorbance at 205 nm and 280 nm using the chromatographic method of Van Iersel et al (1985). Purified MDH (0.7 nmol) was injected on a Superose 12 gel-filtration column equilibrated with 100 mM glycine/ KOH, pH 9.5 (buffer A), plus 5 mM MgSO,, equilibrated at a flow rate of 0.5 ml/min (at room temperature).…”

Section: Absorption Coefficients Of Mdh and Activator Proteinmentioning

confidence: 99%

Properties of an NAD(H)‐Containing Methanol Dehydrogenase and its Activator Protein from Bacillus methanolicus

Arfman

Hektor

Bystrykh

et al. 1997

European Journal of Biochemistry

View full text Add to dashboard Cite

Oxidation of C, -C, primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43 000-M, subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Znz+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mgz+ ions, MDH interacts with a previously identified 50 00044, activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.

show abstract

Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection

Cited by 81 publications

References 15 publications

Inhibition of neutrophil elastase by the α₁‐proteinase inhibitor‐immunoglobulin A complex

Inhibition of neutrophil elastase by the α₁‐proteinase inhibitor‐immunoglobulin A complex

Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Methylophaga marina

Properties of an NAD(H)‐Containing Methanol Dehydrogenase and its Activator Protein from Bacillus methanolicus

Contact Info

Product

Resources

About

Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection

Cited by 81 publications

References 15 publications

Inhibition of neutrophil elastase by the α1‐proteinase inhibitor‐immunoglobulin A complex

Inhibition of neutrophil elastase by the α1‐proteinase inhibitor‐immunoglobulin A complex

Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Methylophaga marina

Properties of an NAD(H)‐Containing Methanol Dehydrogenase and its Activator Protein from Bacillus methanolicus

Contact Info

Product

Resources

About

Inhibition of neutrophil elastase by the α₁‐proteinase inhibitor‐immunoglobulin A complex

Inhibition of neutrophil elastase by the α₁‐proteinase inhibitor‐immunoglobulin A complex