[30]
CYTOCHROMEP450 TURNOVER
315
Buffer will enter the sites of application by capillary action and focus the samples. As an alternative to thin-layer electrophoresis, thin-layer chromatography methods can be applied to resolve and identify phosphoamino acids. 28 Trypsin digestion of P450 should be carried out under two or three conditions (e.g., varying the protease-to-P450 ratio) to ensure complete digestion and thereby avoid misinterpretation of the tryptic maps. Peptide maps of samples prepared in parallel may be compared to ascertain whether the site on the P450 that is phosphorylated in vitro is the same as that which is phosphorylated in intact hepatocytes. As an alternative to thin-layer electrophoresis, phosphorylated P450 peptides may be resolved by high-performance liquid chromatography (HPLC) using standard methods. 29 Acid hydrolysis of tryptic digests under the recommended conditions will typically yield 32p-labeled hydrolyzed peptides, phosphoamino acids, and free phosphate in a ratio of 1 : 1 : 1. P450 phosphorylations characterized to date include serine (rat P450IIB 1 and rabbit P450IIB4) 4,23 and threonine residues 3° as the site of phosphorylation. A cAMP-dependent protein kinase recognition sequence Arg-Arg-X-Ser is present in the region of amino acids 125-130 in many members of P450 gene family II, but it is recognized as a substrate for cAMP-dependent kinases in only some of the P450s that contain this conserved sequence) Acknowledgments Supported in part by Grant DK-33765from the National Institutes of Health (D.J.W.) E. Neufeld, H. J. Goren, and D. Boland, Anal. Biochem. 177, 138 (1989). J. E. Shively, "Methods of Protein Microcharactedzation." Humana Press, Clifton, New Jersey, 1986. 3oI. Vilgrain, G. DeFaye, and E. M. Chambaz, Biochem. Biophys. Res. Commun. 125, 554 (1984).
[30] C y t o c h r o m e P 4 5 0 T u r n o v e r B y MARIA ALMIRA CORREIA
Introduction The hepatic microsomal hemoproteins, collectively termed as cytochrome P450, consist of a family of multiple isozymes, all of which recruit heme (iron-protoporphyrin IX), as their essential prosthetic moiety. METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
316
REGULATORYPARADIGMS
[30]
These hemoproteins are monomeric (MW ~50,000), containing one heme moiety per mole of enzyme. The iron of the heme moiety is coordinated to the thiol ofa cysteine residue located in an invariable and highly conserved pentadecapeptide region (corresponding to Phe-350-Cys-357-Arg-364 of helix L of P450cam) in the COOH terminus of the apocytochrome.1 In P450cam, the thiolate moiety of Cys-357 is believed to be protected from facile oxidation in the external milieu by sequestration in a surface pocket contoured by Phe-350, Leu-358, and Gin-360, residues found to be highly conserved in corresponding eukaryotic P450s. ! Site-directed mutagenesis studies indeed confirm the importance of this naturally designed heme-apoprotein binding pocket. 2 In spite of this protective pocket, dynamic exchange of heme between various apocytochromes o c c u r s 3 and contributes to the normally observed asynchronous turnover of these P450 moieties. In the strictest sense, turnover of any protein inherently is a function of both protein synthesis (a zero-order process) and protein degradation (a first-order rate process). 4 In view of the kinetic nature of these processes, the rate constant of degradation frequently is the sole determinant of t h e " steady-state" concentration of each protein as it oscillates between the basal and the induced/repressed state. However, because the P450 hemoproteins are heterogenous in character, their turnover is a function of the degradation of each constitutive moiety. With the possible exception of a few P450s such as P450IIAI, 5 the turnover of each P450 moiety appears to be essentially monophasic and to conform to a first-order rate process. 5-~°From the apparent first-order rate constants of such a process, the useful turnover parameter of half-life or t~/2 of each moiety may be derived. 1 T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195, 687 (1987). 2 T. Shimizu, K. Hirano, M. Takahashi, M. Hatano, and Y. Fujii-Kuriyama, Biochemistry 27, 4138 (1988). 3 H. Sadano and T. Omura, Biochem. Biophys. Res. Commun. 116, 1013 (1983). 4 j. C. Waterlow, P. J. Garlick, and D. J. Millward, "Protein Turnover in Mammalian Tissues and in the Whole Body," p. 481. North-Holland, Amsterdam, 1978. 5 A. Parkinson, P. E. Thomas, D. Ryan, and W. Levin, Arch. Biochem. Biophys. 225, 216 (1983). 6 H. Shiraki and F. P. Guengerich, Arch. Biochem. Biophys. 235, 86 (1984). 7 H. Sadano and T. Omura, J. Biochem. (Tokyo) 93, 1375 (1983). s R. Gasser, H. P. Hauri, and U. A. Meyer, FEBS Lett. 147, 239 (1982). 9 A. Kumar, H. Ravishankar, and G. Padmanaban, in "Biochemistry, Biophysics and Regulation of Cytochrome P450" O. A. Gustafsson, J. Carlstedt-Duke, A. Mode, and J. Rafter, eds.), p. 423. Elsevier/North-Holland, Amsterdam, 1980. l0 p. B. Watkins, S. A. Wrighton, E. G. Schuetz, P. Maurel, and P. S. Guzelian, J. Biol. Chem. 261, 6264 (1986).
[30]
CYTOCHROMEP450 TURNOVER
317
In general, the P450 heme moieties appear to turn over more rapidly than their corresponding apocytochromes (Table I). Their relatively shorter half-lives may reflect their propensity for ready exchange with the prosthetic heme of other hepatic hemoproteins.3 The apoprotein half-lives of the various P450s vary significantly from each other and other hepatic proteins (Tables I and II), and are considerably shorter than those of the other microsomal incumbents, flavoprotein NADPH-cytochrome-P450 reductase and cytochrome bs. Such differential turnover reinforces the notion that the physiologic turnover of P450s, like that of other cellular proteins, might be a highly selective event. Although, in common with other hepatic hemoproteins, namely, tryptophan pyrrolase (2,3-dioxygenase) n and catalase, ~2the proteolytic turnover of some P450s is accelerated on deprivation of their prosthetic heine, this may be strictly due to acute hepatic heme depletion. Normally, mere loss of prosthetic heme should not lead to proteolysis, because, given their propensity for heme, P450s could readily replace heme from the "free" heme pool under conditions of unimpaired hepatic heme regulation. However, very little is currently known about the precise structural alteration of P450 apoproteins which target them for normal proteolytic turnover and the physiologic processes that regulate such turnover. Nonetheless, it is obvious that factors which influence the synthesis and degradation of each constitutive moiety would control the normal turnover of P450 hemoproteins. Empirical approaches to the rigorous determination of the turnover of each individual P450 thus require a fundamental understanding of these critical controlling factors, if the half-life (t~/2) parameters derived for each constitutive P450 moiety are to be truly representative of their "real" biological turnover and therefore meaningful. Thus, turnover studies in animals pretreated with agents ("inducers") that stabilize certain P450s may not afford the true or intrinsic half-life, but the stabilized or "apparent t~/2" of these hemoproteins. However, determination of such relative "apparent" turnover might suffice when P450 inducers, stabilizers, or inactivators are comparatively evaluated. In such instances, less rigorous experimental approaches may provide the desired information while conserving time, energy, and research costs. The various approaches used in the past for assessment of P450 turnover and their relative merits and demerits have been superbly discussed by Watkins et a l ) 3 Because space constraints do not permit such a discus11 R. T. Schimke, E. W. Sweeney, and C. M. Berlin, J. Biol. Chem. 240, 322 (1965). 12 V. E. Price, W. R. Sterling, V, A. Tarantola, R. W. Hartley, Jr., and M. Rechcigl, Jr., J. Biol. Chem. 240, 322 (1965). 13 p. B. Watldns, J. S. Bond, and P. S. Guzelian, in "Mammalian Cytochromes P450" (F. P. Guengerich, ed.), Vol. 2, p. 173. CRC Press, Boca Raton, Florida, 1987.
o
! o
r-
ul U
. . . .
l:1
z
e~ Z
.( eq
0
m
.=
Z ,.~ m
m
.g N
8
+ l ÷ l ~ + l ÷ l + l + l ~ ÷ l +÷l ~ + l + l ÷~l +ml ~ + l +~l
0
m ~T
+~1 I ~+~l+'l
I+ll-~l
I I+~ I+~1
I I I+11
0
d +~
I I--
÷~
I+1 I I+1 I~
I I I +1 I +~ I I I I ÷~ I
,...I u.l
= e~ z z
e~
=-
[30]
CYTOCHROME P450 TURNOVER
319
TABLE II RELATIVE HALF-LIVES OF LIVER PROTEINS IN UNTREATED RATS
t~/2 (hr) Hemoprotein/enzyme Cytochrome b5 Catalase P450IICII(h) Tryptophan 2,3-dioxygenase NADPH-cytochrome P450 Reductase Epoxide hydrolase Total microsomal protein
K. b R. c B. d H. e R. fH.
Protein
Heme
Ref.
84 60 60 20 +- 3 2.2 29 --- 1 35 19 --- 2 20 - 1 35
41 55 43 19 +- 2 ----15 -+ 1 20
Bock and Siekevitz, 1970a Druyan et al., 1969b Poole et al., 1969c Shiraki and Guengerich, 1984a Schimke et al., 1965e Shiraki and Guengerich, 1984b Sadano and Omura, 1983r Shiraki and Guengerich, 1984d Shiraki and Guengerich, 1984d Sadano and Omura, 1983f
W. Bock and P. Siekevitz, Biochem. Biophys. Res. Commun. 41, 374 (1970). Druyan, B. De Bernard, and M. Rabinowitz, J. Biol. Chem. 244, 5874 (1969). Poole, F. Leighton, and C. De Duve, J. Cell Biol. 41, 536 (1969). Shiraki and F. P. Guengerich, Arch. Biochem. Biophys. 2,35, 86 (1984). T. Schimke, E. W. Sweeney, and C. M. Berlin, J. Biol. Chem. 240, 322 (1965). Sadano and T. Omura, J. Biochem. (Tokyo) 93, 1375 (1983).
sion, the reader is referred to that review for a more comprehensive appreciation of the topic. With these caveats and within the confines of presently evaluated methodology, the discussion that follows strictly applies to technical approaches for assessment of the "true" biological turnover of individual moieties of the rat liver P450s and their potential pitfalls. For starters, the choice of experimental models is of paramount importance and worthy of consideration. Experimental Models for Assessment of "Normal" Rat Liver P 4 5 0 Turnover
The reported ranges of half-lives of some P450s and the well-recognized limited viability (
CYTOCHROMEP450 TURNOVER
315
Buffer will enter the sites of application by capillary action and focus the samples. As an alternative to thin-layer electrophoresis, thin-layer chromatography methods can be applied to resolve and identify phosphoamino acids. 28 Trypsin digestion of P450 should be carried out under two or three conditions (e.g., varying the protease-to-P450 ratio) to ensure complete digestion and thereby avoid misinterpretation of the tryptic maps. Peptide maps of samples prepared in parallel may be compared to ascertain whether the site on the P450 that is phosphorylated in vitro is the same as that which is phosphorylated in intact hepatocytes. As an alternative to thin-layer electrophoresis, phosphorylated P450 peptides may be resolved by high-performance liquid chromatography (HPLC) using standard methods. 29 Acid hydrolysis of tryptic digests under the recommended conditions will typically yield 32p-labeled hydrolyzed peptides, phosphoamino acids, and free phosphate in a ratio of 1 : 1 : 1. P450 phosphorylations characterized to date include serine (rat P450IIB 1 and rabbit P450IIB4) 4,23 and threonine residues 3° as the site of phosphorylation. A cAMP-dependent protein kinase recognition sequence Arg-Arg-X-Ser is present in the region of amino acids 125-130 in many members of P450 gene family II, but it is recognized as a substrate for cAMP-dependent kinases in only some of the P450s that contain this conserved sequence) Acknowledgments Supported in part by Grant DK-33765from the National Institutes of Health (D.J.W.) E. Neufeld, H. J. Goren, and D. Boland, Anal. Biochem. 177, 138 (1989). J. E. Shively, "Methods of Protein Microcharactedzation." Humana Press, Clifton, New Jersey, 1986. 3oI. Vilgrain, G. DeFaye, and E. M. Chambaz, Biochem. Biophys. Res. Commun. 125, 554 (1984).
[30] C y t o c h r o m e P 4 5 0 T u r n o v e r B y MARIA ALMIRA CORREIA
Introduction The hepatic microsomal hemoproteins, collectively termed as cytochrome P450, consist of a family of multiple isozymes, all of which recruit heme (iron-protoporphyrin IX), as their essential prosthetic moiety. METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
316
REGULATORYPARADIGMS
[30]
These hemoproteins are monomeric (MW ~50,000), containing one heme moiety per mole of enzyme. The iron of the heme moiety is coordinated to the thiol ofa cysteine residue located in an invariable and highly conserved pentadecapeptide region (corresponding to Phe-350-Cys-357-Arg-364 of helix L of P450cam) in the COOH terminus of the apocytochrome.1 In P450cam, the thiolate moiety of Cys-357 is believed to be protected from facile oxidation in the external milieu by sequestration in a surface pocket contoured by Phe-350, Leu-358, and Gin-360, residues found to be highly conserved in corresponding eukaryotic P450s. ! Site-directed mutagenesis studies indeed confirm the importance of this naturally designed heme-apoprotein binding pocket. 2 In spite of this protective pocket, dynamic exchange of heme between various apocytochromes o c c u r s 3 and contributes to the normally observed asynchronous turnover of these P450 moieties. In the strictest sense, turnover of any protein inherently is a function of both protein synthesis (a zero-order process) and protein degradation (a first-order rate process). 4 In view of the kinetic nature of these processes, the rate constant of degradation frequently is the sole determinant of t h e " steady-state" concentration of each protein as it oscillates between the basal and the induced/repressed state. However, because the P450 hemoproteins are heterogenous in character, their turnover is a function of the degradation of each constitutive moiety. With the possible exception of a few P450s such as P450IIAI, 5 the turnover of each P450 moiety appears to be essentially monophasic and to conform to a first-order rate process. 5-~°From the apparent first-order rate constants of such a process, the useful turnover parameter of half-life or t~/2 of each moiety may be derived. 1 T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195, 687 (1987). 2 T. Shimizu, K. Hirano, M. Takahashi, M. Hatano, and Y. Fujii-Kuriyama, Biochemistry 27, 4138 (1988). 3 H. Sadano and T. Omura, Biochem. Biophys. Res. Commun. 116, 1013 (1983). 4 j. C. Waterlow, P. J. Garlick, and D. J. Millward, "Protein Turnover in Mammalian Tissues and in the Whole Body," p. 481. North-Holland, Amsterdam, 1978. 5 A. Parkinson, P. E. Thomas, D. Ryan, and W. Levin, Arch. Biochem. Biophys. 225, 216 (1983). 6 H. Shiraki and F. P. Guengerich, Arch. Biochem. Biophys. 235, 86 (1984). 7 H. Sadano and T. Omura, J. Biochem. (Tokyo) 93, 1375 (1983). s R. Gasser, H. P. Hauri, and U. A. Meyer, FEBS Lett. 147, 239 (1982). 9 A. Kumar, H. Ravishankar, and G. Padmanaban, in "Biochemistry, Biophysics and Regulation of Cytochrome P450" O. A. Gustafsson, J. Carlstedt-Duke, A. Mode, and J. Rafter, eds.), p. 423. Elsevier/North-Holland, Amsterdam, 1980. l0 p. B. Watkins, S. A. Wrighton, E. G. Schuetz, P. Maurel, and P. S. Guzelian, J. Biol. Chem. 261, 6264 (1986).
[30]
CYTOCHROMEP450 TURNOVER
317
In general, the P450 heme moieties appear to turn over more rapidly than their corresponding apocytochromes (Table I). Their relatively shorter half-lives may reflect their propensity for ready exchange with the prosthetic heme of other hepatic hemoproteins.3 The apoprotein half-lives of the various P450s vary significantly from each other and other hepatic proteins (Tables I and II), and are considerably shorter than those of the other microsomal incumbents, flavoprotein NADPH-cytochrome-P450 reductase and cytochrome bs. Such differential turnover reinforces the notion that the physiologic turnover of P450s, like that of other cellular proteins, might be a highly selective event. Although, in common with other hepatic hemoproteins, namely, tryptophan pyrrolase (2,3-dioxygenase) n and catalase, ~2the proteolytic turnover of some P450s is accelerated on deprivation of their prosthetic heine, this may be strictly due to acute hepatic heme depletion. Normally, mere loss of prosthetic heme should not lead to proteolysis, because, given their propensity for heme, P450s could readily replace heme from the "free" heme pool under conditions of unimpaired hepatic heme regulation. However, very little is currently known about the precise structural alteration of P450 apoproteins which target them for normal proteolytic turnover and the physiologic processes that regulate such turnover. Nonetheless, it is obvious that factors which influence the synthesis and degradation of each constitutive moiety would control the normal turnover of P450 hemoproteins. Empirical approaches to the rigorous determination of the turnover of each individual P450 thus require a fundamental understanding of these critical controlling factors, if the half-life (t~/2) parameters derived for each constitutive P450 moiety are to be truly representative of their "real" biological turnover and therefore meaningful. Thus, turnover studies in animals pretreated with agents ("inducers") that stabilize certain P450s may not afford the true or intrinsic half-life, but the stabilized or "apparent t~/2" of these hemoproteins. However, determination of such relative "apparent" turnover might suffice when P450 inducers, stabilizers, or inactivators are comparatively evaluated. In such instances, less rigorous experimental approaches may provide the desired information while conserving time, energy, and research costs. The various approaches used in the past for assessment of P450 turnover and their relative merits and demerits have been superbly discussed by Watkins et a l ) 3 Because space constraints do not permit such a discus11 R. T. Schimke, E. W. Sweeney, and C. M. Berlin, J. Biol. Chem. 240, 322 (1965). 12 V. E. Price, W. R. Sterling, V, A. Tarantola, R. W. Hartley, Jr., and M. Rechcigl, Jr., J. Biol. Chem. 240, 322 (1965). 13 p. B. Watldns, J. S. Bond, and P. S. Guzelian, in "Mammalian Cytochromes P450" (F. P. Guengerich, ed.), Vol. 2, p. 173. CRC Press, Boca Raton, Florida, 1987.
o
! o
r-
ul U
. . . .
l:1
z
e~ Z
.( eq
0
m
.=
Z ,.~ m
m
.g N
8
+ l ÷ l ~ + l ÷ l + l + l ~ ÷ l +÷l ~ + l + l ÷~l +ml ~ + l +~l
0
m ~T
+~1 I ~+~l+'l
I+ll-~l
I I+~ I+~1
I I I+11
0
d +~
I I--
÷~
I+1 I I+1 I~
I I I +1 I +~ I I I I ÷~ I
,...I u.l
= e~ z z
e~
=-
[30]
CYTOCHROME P450 TURNOVER
319
TABLE II RELATIVE HALF-LIVES OF LIVER PROTEINS IN UNTREATED RATS
t~/2 (hr) Hemoprotein/enzyme Cytochrome b5 Catalase P450IICII(h) Tryptophan 2,3-dioxygenase NADPH-cytochrome P450 Reductase Epoxide hydrolase Total microsomal protein
K. b R. c B. d H. e R. fH.
Protein
Heme
Ref.
84 60 60 20 +- 3 2.2 29 --- 1 35 19 --- 2 20 - 1 35
41 55 43 19 +- 2 ----15 -+ 1 20
Bock and Siekevitz, 1970a Druyan et al., 1969b Poole et al., 1969c Shiraki and Guengerich, 1984a Schimke et al., 1965e Shiraki and Guengerich, 1984b Sadano and Omura, 1983r Shiraki and Guengerich, 1984d Shiraki and Guengerich, 1984d Sadano and Omura, 1983f
W. Bock and P. Siekevitz, Biochem. Biophys. Res. Commun. 41, 374 (1970). Druyan, B. De Bernard, and M. Rabinowitz, J. Biol. Chem. 244, 5874 (1969). Poole, F. Leighton, and C. De Duve, J. Cell Biol. 41, 536 (1969). Shiraki and F. P. Guengerich, Arch. Biochem. Biophys. 2,35, 86 (1984). T. Schimke, E. W. Sweeney, and C. M. Berlin, J. Biol. Chem. 240, 322 (1965). Sadano and T. Omura, J. Biochem. (Tokyo) 93, 1375 (1983).
sion, the reader is referred to that review for a more comprehensive appreciation of the topic. With these caveats and within the confines of presently evaluated methodology, the discussion that follows strictly applies to technical approaches for assessment of the "true" biological turnover of individual moieties of the rat liver P450s and their potential pitfalls. For starters, the choice of experimental models is of paramount importance and worthy of consideration. Experimental Models for Assessment of "Normal" Rat Liver P 4 5 0 Turnover
The reported ranges of half-lives of some P450s and the well-recognized limited viability (
