- Email: [email protected]
Cytochromes of Bacillus megaterium and Bacillus subtilis
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Cytochromes
103-104 (1960)
90,
of Bacillus Bacillus
I’rom the Brigham
megaterium
subtili5’
Young University,
Received
and
March
Proz~o, I:tah
21, 1960
Treatment of Bacillus snblilis and Bacillus meyaterium cells by ordinary procedures failed to ext,ract any soluble cytochrome from the cell. Sonoration produced small fragments which contained the cytochrome complement and were sedimentable in a centrifugal field of 144,000 X q. Difference spectra of such part,icles showed absorption maxima at 599, 557, 552, 530, and 428 mr. ljigestion of the fragments with lipase liberated a rather typical cytochrome c in that, it had absorption maxima in the reduced form at. 550, 520, and 415 rnp, formed cyanide and pyridine hemochromogew which w-ere spectroscopically similar to those obtained from mammalian cytochrome r, could be purified in the usual manner by ammonium sulfate fractionation, and had an oxidation potential at, pH 7.0 of 0.25 v. As isolated, however, the cptjochrome c was rendil,v oxidized by oxygen in the air.
part,icle which was sedimentable at low centrifugal fields and cont’ained all the cytochromes. Extraction of acetone-dried powders or treatmentj of cells with warm trichloroacetic or citric acid did not liberat,c any c?ochrome. Sonic oscillation of cells for 30 mm. in a IO kc. Raytheon Magnetorestrictor produced small cell fragmenk which mere not sedimented by centrifugation at 60,000 X y. After au initial centrifugation at 60,000 X g to remove cell debris, a suhsequent centrifugation at 144,000 X g for 30 min. produced a small pellet and also a cow centration of pigment in t,he fluid immediately above t’he pellet’. Figure 1 presents a difference spectrum of such a “color layer” from B. mrgaterium. This spectrum is similar t,o difference specka obtained using either t)he rcsuspwded partjicles from t,he pellet OI the origiual cxtractj made by sonic oscillatiou. A11 these fra&ions showed maxima in their diffcrenw spectra at 399, 337, 552, 530, and 423 nip. B. s2dAili.s cells gave spcct,roscopitally knilar fractions. All the data indicate that the cytochromes in these bacterial ~11s were cwkained on some particulat,c st,ructure, and the elegant work of W&bull and Bergst,rom (5) show that’ the structure coutaining the particles n-as the cell membrane.
011 whole Bacillus su6tilis cells have shown the presence of a cytochrome system similar to that found in mammalian tjissucs (l-4). Working with a closelr related organism, Bacillus megaterium, Weibull and Bergstrom reported a similar cyt’ochrome composition for t)his organism and further demonst,ratcd t’hat t,he cytochromes were cont,ained in the cell membrane (5). This commui~i~at~ion concerns an iiivestigation of the cytochrome composition of B. s7&ilis (University of Wisconsin collection) and B. megatrrium (a st,raiu isolated in the laboratory of Dr. ,J. V. Beck). The cytochrome compositiou of these bacteria corresponds to tjhat previously reported (l-5)) and a cytochrome c has been partially purified from cells of each bacterial species. Investigations
I‘se of a French cell or alumina grinding to rupture the bact,erial cells produced a 1 This invcstigat.ion was supported by a rcscnwh grant (ljo. E-917C) from the National Inatitlite of Allergy and Infectious l)iseases, Sxtional 1rrstitlltr.q of Health, U. S. Public Health Service. 9 Prrsent address: I)epartment of Hiochemistry, T7nivcrsity of Washington, Seattle, Washington. 103
104 r
I
I
L I
Wove
I
Length
rnf
FIG. 1. Difference spectrnm (reduced minw oxidized) of particles from R. meyaterium which concentrated above pellet following centrifngation for 30 min. at 114,000 X 0. The spectrum wax obtained with a Beckman DK2 recording spectrophotometer.
400
425
4.50
475 300 550 wove LengN,nlfl
600
700
FIG. 2. The reduced, direct spectrum of pnrified qtochrome c from 13. ,7,eyate,.izrn2.
Them was considerable amounts of cytochromc b in the “color layer” from some cases cytochrome c
variation in the and cytochrome c these backria. In was predominant,
l&h little or no cytochrome 11, and \-ice versa. The cyt,ochrome c from both B. ?wq~~tcrium and B. .suOtilis was contained in t,he particles derived from t,he cell membrane and was not released from the particle 1)~ any of the procedures ordinarily used to cxtr& cytochromc (8from bacterial or mammalian cells. The vytochrome c was released, however, by digestion with lipase at room temperature for 12 hr. The cytochromc ( liberated by this treatment was typical ill that it could be purified by ammonium sulfate frnctiouation (precipitating at, high salt concentration in the presence of trichloroacet,ic acid) and had an absorption spectrum similar to mammalian cytochrome (2 (6). The absorption spectrum of the reduced cytochrome c from B. megatuivcm is sho\vn in Fig. 2. As isolated, this cytochromc had absorption maxima at, 550, 520, and 413 rnp in the reduced form, reacted wit’h vyanidc and pyridine to give reduced hemochromogens with spect,roscopic properties similar to mammalian cytochromc c (6)) was readily reoxidizcd by t)he oxygen in air, and had a potential of 0.25 v. at pH 7.0. The ease of oxidation in air could be due to the lipase digestion, and need not reflect the condition of the cytochrome as it occurred in the particle derived from the cell membrane. X cytochromc c from B. su6tilis was prepared using the lipase treatment to release t’hr \VLlS and t)his cyt’ochrome cyt,ochrome, spectroscopically identical in t#he visible range wit,h t,he cytochrome c from B. mqaterium. In all experiments performed to date itI has not been possible to remove the cytochrome a or cyt,ochromc b from the particles of either hact,erial specsics, and uo purification of these cytochromes has been possible.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Cytochromes
103-104 (1960)
90,
of Bacillus Bacillus
I’rom the Brigham
megaterium
subtili5’
Young University,
Received
and
March
Proz~o, I:tah
21, 1960
Treatment of Bacillus snblilis and Bacillus meyaterium cells by ordinary procedures failed to ext,ract any soluble cytochrome from the cell. Sonoration produced small fragments which contained the cytochrome complement and were sedimentable in a centrifugal field of 144,000 X q. Difference spectra of such part,icles showed absorption maxima at 599, 557, 552, 530, and 428 mr. ljigestion of the fragments with lipase liberated a rather typical cytochrome c in that, it had absorption maxima in the reduced form at. 550, 520, and 415 rnp, formed cyanide and pyridine hemochromogew which w-ere spectroscopically similar to those obtained from mammalian cytochrome r, could be purified in the usual manner by ammonium sulfate fractionation, and had an oxidation potential at, pH 7.0 of 0.25 v. As isolated, however, the cptjochrome c was rendil,v oxidized by oxygen in the air.
part,icle which was sedimentable at low centrifugal fields and cont’ained all the cytochromes. Extraction of acetone-dried powders or treatmentj of cells with warm trichloroacetic or citric acid did not liberat,c any c?ochrome. Sonic oscillation of cells for 30 mm. in a IO kc. Raytheon Magnetorestrictor produced small cell fragmenk which mere not sedimented by centrifugation at 60,000 X y. After au initial centrifugation at 60,000 X g to remove cell debris, a suhsequent centrifugation at 144,000 X g for 30 min. produced a small pellet and also a cow centration of pigment in t,he fluid immediately above t’he pellet’. Figure 1 presents a difference spectrum of such a “color layer” from B. mrgaterium. This spectrum is similar t,o difference specka obtained using either t)he rcsuspwded partjicles from t,he pellet OI the origiual cxtractj made by sonic oscillatiou. A11 these fra&ions showed maxima in their diffcrenw spectra at 399, 337, 552, 530, and 423 nip. B. s2dAili.s cells gave spcct,roscopitally knilar fractions. All the data indicate that the cytochromes in these bacterial ~11s were cwkained on some particulat,c st,ructure, and the elegant work of W&bull and Bergst,rom (5) show that’ the structure coutaining the particles n-as the cell membrane.
011 whole Bacillus su6tilis cells have shown the presence of a cytochrome system similar to that found in mammalian tjissucs (l-4). Working with a closelr related organism, Bacillus megaterium, Weibull and Bergstrom reported a similar cyt’ochrome composition for t)his organism and further demonst,ratcd t’hat t,he cytochromes were cont,ained in the cell membrane (5). This commui~i~at~ion concerns an iiivestigation of the cytochrome composition of B. s7&ilis (University of Wisconsin collection) and B. megatrrium (a st,raiu isolated in the laboratory of Dr. ,J. V. Beck). The cytochrome compositiou of these bacteria corresponds to tjhat previously reported (l-5)) and a cytochrome c has been partially purified from cells of each bacterial species. Investigations
I‘se of a French cell or alumina grinding to rupture the bact,erial cells produced a 1 This invcstigat.ion was supported by a rcscnwh grant (ljo. E-917C) from the National Inatitlite of Allergy and Infectious l)iseases, Sxtional 1rrstitlltr.q of Health, U. S. Public Health Service. 9 Prrsent address: I)epartment of Hiochemistry, T7nivcrsity of Washington, Seattle, Washington. 103
104 r
I
I
L I
Wove
I
Length
rnf
FIG. 1. Difference spectrnm (reduced minw oxidized) of particles from R. meyaterium which concentrated above pellet following centrifngation for 30 min. at 114,000 X 0. The spectrum wax obtained with a Beckman DK2 recording spectrophotometer.
400
425
4.50
475 300 550 wove LengN,nlfl
600
700
FIG. 2. The reduced, direct spectrum of pnrified qtochrome c from 13. ,7,eyate,.izrn2.
Them was considerable amounts of cytochromc b in the “color layer” from some cases cytochrome c
variation in the and cytochrome c these backria. In was predominant,
l&h little or no cytochrome 11, and \-ice versa. The cyt,ochrome c from both B. ?wq~~tcrium and B. .suOtilis was contained in t,he particles derived from t,he cell membrane and was not released from the particle 1)~ any of the procedures ordinarily used to cxtr& cytochromc (8from bacterial or mammalian cells. The vytochrome c was released, however, by digestion with lipase at room temperature for 12 hr. The cytochromc ( liberated by this treatment was typical ill that it could be purified by ammonium sulfate frnctiouation (precipitating at, high salt concentration in the presence of trichloroacet,ic acid) and had an absorption spectrum similar to mammalian cytochrome (2 (6). The absorption spectrum of the reduced cytochrome c from B. megatuivcm is sho\vn in Fig. 2. As isolated, this cytochromc had absorption maxima at, 550, 520, and 413 rnp in the reduced form, reacted wit’h vyanidc and pyridine to give reduced hemochromogens with spect,roscopic properties similar to mammalian cytochromc c (6)) was readily reoxidizcd by t)he oxygen in air, and had a potential of 0.25 v. at pH 7.0. The ease of oxidation in air could be due to the lipase digestion, and need not reflect the condition of the cytochrome as it occurred in the particle derived from the cell membrane. X cytochromc c from B. su6tilis was prepared using the lipase treatment to release t’hr \VLlS and t)his cyt’ochrome cyt,ochrome, spectroscopically identical in t#he visible range wit,h t,he cytochrome c from B. mqaterium. In all experiments performed to date itI has not been possible to remove the cytochrome a or cyt,ochromc b from the particles of either hact,erial specsics, and uo purification of these cytochromes has been possible.