Purification of cytochrome b-561

Silsand, Terje; Flatmark, Torgeir

doi:10.1016/0005-2795(74)90223-2

Cited by 42 publications

(9 citation statements)

References 20 publications

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“…Visible Absorption Spectra-The purified cytochrome b 561 in reduced form showed ␣, ␤, and Soret absorption maxima at 561, 530, and 427 nm, respectively, as reported previously (5). At the final stage of the purification, sodium ascorbate was omitted from the elution buffer to remove the absorption in ultraviolet region due to ascorbic acid or semidehydroascorbic acid.…”

Section: Purification Of Cytochrome B 561 -supporting

confidence: 64%

“…Its amino acid sequence shows no apparent homology with any other membrane-bound cytochromes so far known (2). The cytochrome is characterized by a rather high redox midpoint potential (4) and an asymmetric absorption peak in the ␣-band with a maximum at 561 nm and a shoulder at approximately 558 nm (5). Apps et al (6) revealed the presence of two potentiometrically different forms (midpoint potentials, 170 and 70 mV, respectively) of cytochrome b 561 and reported the high and low potential components to have identical absorption spectra.…”

mentioning

confidence: 99%

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Existence of Two Heme B Centers in Cytochromeb 561 from Bovine Adrenal Chromaffin Vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy

Tsubaki¹,

Nakayama²,

Okuyama³

et al. 1997

Journal of Biological Chemistry

100

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We have established a new purification procedure of cytochrome b 561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochrome b 561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at g z ‫؍‬ 3.14 and g z ‫؍‬ 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at g z ‫؍‬ 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochrome b (b L or b 566 ) of the mitochondrial complex III, indicating that the g z ‫؍‬ 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid-and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.

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Section: Purification Of Cytochrome B 561 -supporting

confidence: 64%

mentioning

confidence: 99%

Existence of Two Heme B Centers in Cytochromeb 561 from Bovine Adrenal Chromaffin Vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy

Tsubaki¹,

Nakayama²,

Okuyama³

et al. 1997

Journal of Biological Chemistry

100

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“…The main contaminants of cmde secretory vesicle fractions (Le., those obtained by differential centrifugation) are mitochondria, which can be completely removed using such gradients. The vesicles obtained are stable and have successfully been used in studies concerned with the analysis of proton, sotium, and calcium transport (6,7,13,20,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

Section: Resul Ts and Discussionmentioning

confidence: 99%

“…The details of the methods used in the assays for five different acid hydrolases (14)(15)(16) are given in Table 1. Cytochrome b S61 was estimated from the reduced minus oxidized difference spectra (17). A TPases were measured as described (18,19).…”

Section: Methodsmentioning

confidence: 99%

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Gratzl

1984

Analytical Biochemistry

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emde chromaffin secretory vesicles, obtained by differential centrifugation, were further purified on isotonic (PereolI) gradients. The chromaffin vesicIe fractions recovered from the gradients contain acetylcholinesterase as well as lysosomal enzymes. With the aid of a subsequent sucrose gradient lysosomal enzymes could be removed from chromaffin vesicIe fractions. but not acetylcholinesterase. This suggests that lysosomal enzymes do not pass through the chromaffin vesicIes during the biogenesis of lysosomes but acetylcholinesterase does.

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“…(DBH) catalyse la derniere Ctape de la synthkse de la noradrknaline par hydroxylation de la dopamine [ 1 ] . L'enzyme est 1ocalisCe au niveau des granules chromaffines des cellules de la m6dullo-surr6nale…”

Section: Introductionunclassified

Proprietes cinetiques de la dopamine‐β‐hydroxylase membranaire

Portugal¹,

Jordá²,

Ruiz³

1976

FEBS Letters

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SUMMARYWe studied membrane bound dopamine-p-hydroxylase (DBH) from chromaffin granules, in order to determine whether a biological form of immobilization of the enzyme altered its kinetic properties. The results obtained suggested that DBH either in soluble or solubilized form showed a ping-pong mechanism whereas the membrane bound DBH did not. Affinity for the substrate and the pH stability were lower in soluble or solubilized form than in membrane bound DBH.

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Purification of cytochrome b-561

Cited by 42 publications

References 20 publications

Existence of Two Heme B Centers in Cytochromeb 561 from Bovine Adrenal Chromaffin Vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy

Existence of Two Heme B Centers in Cytochromeb 561 from Bovine Adrenal Chromaffin Vesicles as Revealed by a New Purification Procedure and EPR Spectroscopy

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Proprietes cinetiques de la dopamine‐β‐hydroxylase membranaire

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