171

ISOENZY MES tions make it possible to ascribe specific roles to particular residues, such as the conserved glycine residues 141at bends in the basic fold which appear to constitute alcohol dchydrogenase per se. Each of these concepts can now be further tested by directed mutagenesis. Regarding the enzymic mechanisms, the basic properties of the zinc dehydrogenases have essentially been known for some time. However, the short-chain dehydrogenase type is largely unknown in mechanistic terms and studics of additional forms, complemented with extensive comparisons, should be valuable also for the short-chain dehydrogenase group, to define more constant segments and particularly conserved residues which can then be directly tested for in mechanistic and functional terms. For example, a segment around position 150 in the short-chain dehydrogenases appears likely to be close to the active site and has a few conserved residues of particular interest 161. It is t o be expected that further studies of natural variants will increase the reliability in the interpretational conclusions. Support by grants from the Swedish Medical Kesearch Council (project 03X-3532) and the Swedish Alcohol Kcwarch Fund i \ gratefully acknowledged. 1, Jiirnvall. H.. Persson. M. & Jeffery. J. i I98 I ) /’roc,. Nhtl. Accicl. .Sc.i. U..S.A.78. 4226-4230

2. Jiirnvall. H.. von Bahr-Lindstriirn. H. 6i Hi)tig. J . 4 . ( I O X V I in Hitrnnn Merciholisrn o$Alco/iol(Crow, K. t.& Batt, R. D., cds.). vol. 11, pp. 43-64. C R C Press. Boca Raton. FA 3. Aronson. B. D.. Sornerville, R. L.. Epperly. 13. R. & Ikkker. E. E. ( 1089j J. Biol. C’lic~m.264, 5226-5232 4. Borras, T.. P e r s o n , B. & Jiirnvall. H. ( 1989) Niochcvni.srn 28. 6 133-61 39 5. Jiirnvall. H.. von Bahr-Lindstriirn. H.. Jany. K.-I>.. Ulnicr. W. 6i Friicchle, M. ( 1984) FELLS LPII.165, 190- I96 6. Krook, M.. Marekov. L. 6i Jiirnvall. H. ( 1090) /lioc.licwii.\tn~29, in the press 7. Baker. M. 1 19891 ,\.lo/. Encloc~irrol.3. X X I -X84 8. Scopes. R. K. i I983 i FEES l-t,/t. 156. 303-306 9. Wiliiamson. V. M. 6i Paquin. C. E. I 9x7) ,\to/. (;cn. ( ; m v . 209,374-38 I 10. Thatcher. D. R. & Sawyer. L. 1 I 9801 H i o c , h c > m .J. 187, 884-886 I I . Kaiser. R.. Holrnquist. B.. Vallcc. B. L. 6i Jornvall. H. ( 19x9) Biochemistn 28.8432-8338 12. Eklund. H.. Horjales. E.. Vallee. B. L. 6i J6rnvall. H. 1987) Ettr. J. E i o c . h ~ ~ t 167, n. 1x5- I93 13. Eklund, H.. Horjales. E.. Jiirnvall. H.. I3riindi.n. C:I. 6i Jcffcry. J. ( 1985) R i ~ c ~ h e 24, ~ i 8005-801 i.~~~ 2 14. Jiirnvall, H.. P e r s o n , B. & Jelfery. J . (19x7) I-rtr. ./. Eiochorn. 167, 195-201 15. Villarroya, A,. Juan. E.. Egestad. B. & Jiirnvall. H. ( I 989) Eur. J. Hiochern. 180, 191-197

Kcceived I 0 October 19x9

Structure and expression of mammalian carbonic anhydrases YVONNE EDWARDS M .R.C’. Hirmuti Riochemicul Getietics Utiit, 7 % Gullon ~ Laboratmy, lJtiiver.sip College Loticloti, Wolf.i.otrIloirse, 4 Stephetisoti Way, Lotidoti NWI X i E , U.K. Carbonic anhydrase (CA). is a ubiquitous enzyme involved in the transport o f COz between metabolizing tissue and the lungs. in many secretory processes. in ion transport and in the provision of bicarbonate f o r fatty acid synthesis, gluconeogenesis and urea-genesis (see 11 ] for a review and references). Despite the diversity of its metabolic involvements. the reaction that this enzyme catalyses is a simple one, namely the transfer of an OH- moiety bctwcen H,O and CO,:

CO, + H > O =HCO; + H It was in the 196Os, with the advent of chromatographic techniques, that the first isoforms of CA were separated from erythrocytes, namely CAI which is present in high amounts, but with low activity, and CAI1 which is present in a smaller quantity, but with high specific activity. In higher vertebrates at least seven genetically distinct, but structurally similar, isoenzymes o f CA are now recognized, each showing characteristic kinetic properties and tissue distribution (see [ 2 ]for a review). Three of the mammalian CA genes are expressed predominantly in one cell type, cytoplasmic CAI and CAW in erythrocytes and skeletal muscle cells, respectively, and the secreted form, CAVI, in parotid salivary gland. These genes show a greater degree o f tissue specificity than the more ubiquitously expressed cytoplasmic CAI1 o r the membraneassociated CAIV and mitochondria1 CAV. It is not clear if any particular cell type expresses a single C A gene most express two or more. So, for example, erythrocytes and skeletal muscle slow-twitch fibres each express CAI, CAI1 and CAIII, although the relative levels of expresAbbreviations used: C‘A. carbonic anhydrase; 1 ,25(OH)2D,. I.25-dihydroxyvitarnin I>.,; GH. pituitary growth hormone.

VOl. 18

sion of the three genes vary considerably between the two tissues with CAI > CAI1 > > CAI11 for erythrocytes and CAIII > CAI1 > > C A I for slow-twitch fibres. The CA isoforms show between 28%)and 59% amino acid sequence similarity when pairwise comparisons are made, with CAI and CAI1 showing greatest similarity. Recently, Tashian [2] has used consensus amino acid sequences to construct a comparative matrix and a phylogenetic branching scheme for the CA isoforms. These data indicate an order of evolutionary relatedness: CAI. CAII, CAIII. CAVII, CAVl and CAIV and suggest that the salivary CAVl gene diverged first from the ancestral root. This pattern of genetic ancestory is reflected in the chromosomal distribution of the mammalian CA genes. CAI, CAI1 and CAlll are clustered o n the long arm of human chromosome 8 at q22 13. 41. and are similarly clustered o n mouse chromosomes near to the centromere IS.61, while the more distantly related CAVl and CAVll have been mapped to human chromosomes 1 and 16, respectively [ 7 , 8 ] . Why is a complex family of several CA genes required t o encode a monomeric enzyme which catalyses a very simple reaction? Comparisons of these genes and their protein products suggest that the diversity is important both in the context of gene expression and protein function. Variation in those parts of the DNA which encode the CA confers characteristic kinetic properties on each of the isoforms and also provides the protein sequences necessary for particular subcellular localizations or secretion of the gene product, while variation in the non-coding regions provides for flexibility of expression among many different cell types. I’roteiti structure

Fig. 1 shows the 36 active-site residues for six CA isoforms, 17 of these are common to all CAs, while others are characteristic of particular isoforms. Insights into the role of such isoform-specific residues in active-site mechanisms are beginning to emerge from site-directed mutagenesis studies. For example, CAI11 shows eight unique residues in all five

172

BIOCHEMICAL SOCIETY TRANSACTIONS ResNdue no

2

6

6

6

7

9

1

2

4

6

5

6

6

6

6

7

9

9

9

1

9

2

C H

1 0

9

4

6

1 0

6

1 1

7

1 1

1 2

7

9

1 3

1

CAI

161

Y

S

N

V

H

S

CAI1

161

Y

S N

N

H

3

CAI11

151

Y

S

CAV

(ti

X

CAVl

(11

Y

S

N

N

H

T

V

Q

S

K

Q

H

H

E

H

E

H

V

Y

CAVII

I11

1

S

N

N

H

S

V

9

D

K

Q

H

H

E

H

E

H

V

F

N S

N [kl T

N

~

f[hl[nl*

1 4

1

1 4

1

1 9

1 9

1 9

1 9

2 0

2 0

2 0

2 0

3

1 4

5

2

4

8

9

0

1

2

4

3;

S

H

E

H

E

H

Y

f

L[iIg

W

Y

L

T[HlP

P

L

L

2 0

2 0

2 0

6

2 1

7

2 4

9

W l i l N R

V

F[IlCEl[ilQ

H

H

E

H

E

H

V

F

V

G

W

Y

T

T

P

P[11c

W

V

N

H

H

E

H

E

H

V

f [I] V

G

W

Y [PI T

T

P

P [El C [I1 Y

L

N

Q

F

~

V

K

~

H

H

E L

L

V V

H A G

E Y W

Y

Y

H L

T

L T

F T

~ P

T

L

P

P

'

P

~

V

[ClCRI V [ R I 9 H

2 4

1

~

~

~

6

R R ~

L

I

N

V

W

V

D

R

S

N

V

U

V

N

R

T

T

P

P

A

Fig. 1. Residues postriluted to occrir wirhiti the uctive sire of the vertehrute curhotiic utihydrases Residue number is based on human CAI sequence; numbers in parentheses indicate the number of species investigated; residues in square brackets are unique to an isoform. At any one position lower case indicates preponderant residue and upper case uniformity; * indicates variable residue. Data are mainly from [2] and also include sheep CAVl [13], mouse and rat CAlll [3Y, 401. CAY is a sequence derived from a mouse liver cDNA (see [2])and it possibly represents CAV.

species analysed. These include the basic residues Arg or Lys at positions 64, 67 and 9 1 and unique Cys-66, Ile- 14 1, Phe198, Glu-204 and lle-207 residues. C A M differs from other cytoplasmic isoenzymes in having a relatively low rate of CO, hydration and kinetic parameters which are independent of pH in the region 5.5-8.0. It is also relatively insensitive to sulphonamide inhibition and has an unusual p-nitrophenylphosphatase activity at low pH. His-64 in other CA isoforms is thought to play a vital role in proton transfer and the possibility that the His-64 Lys substitution in CAI11 could account for the low activity of this isoform has been investigated. Forsman er ul. [9] replaced His-64 with Lys in the high-activity CAI1 by site-directed mutation. This substitution did not significantly alter the rate of CO, hydration. Similarly, Tu er ul. [ 101 found that the replacement of Lys-64 by His and Arg-67 by Asn in human CAlll does not have a large effect on the rate of C0,-HCO, interconversion, suggesting that these residues may not be responsible for the lower catalytic activity of CAIII. However, crystollographic studies [ l l ] show that thc active-site centre of human CAI11 is more sterically constrained than that of the high activity CAI1 owing to the Phe- 198. Ile-207 and is more positively charged owing to Lys-64 and Arg-67, and this limits the access of small molecule buffers to the zinc-bound hydroxide. Substitution of Arg-67 with Asn or His in C A M significantly increases the access to buffer cations and increases the rate of transfer of the proton between the buffer and the zinc-bound hydroxide. and thus the rate of release of H 2 0from the active site [lo]. Other striking structural differences distinguish the noncytoplasmic CA isoenzymes (CAIV, V and VI) from the cytoplasmic forms. These features can largely be ascribed to the presence of 'topogenic' sequences necessary to achieve correct membrane integration, targeting to a subcellular particle, and secretion. There is, for example, some evidence for cleavable signal sequences on both CAV and CAVI. CAV is targeted to the mitochondria1 matrix presumably by a leader sequence which is subsequently cleaved. Analysis of the N-terminal amino acid sequence of CAV purified from guinea-pig liver mitochondria shows 33-46% identity with the cytosolic isoenzymes, but the loss of sequence similar to residues 1-21 [ 121. This suggests that the post-translational cleavage of the signal sequence occurs between residues corresponding to 2 1 and 22 in other CAs (Fig. 2). CAVI is a glycoprotein secreted by parotid salivary glands. The human CAIV cDNA shows a cleavable signal sequence encoded at the N-terminus which is not present in the mature protein [13]. The processed protein comprises 307 amino acids, compared with the 259 or 260 residues of the cyto-

-

plasmic CAs, with most of the extra sequence contained in a hydrophilic C-terminal extension. No sequence data have yet been published for the membrane-associated CAW. However, the lung and kidney microsomal forms have apparent M , values of 52000 and 68 000, respectively, and contain at least 20% carbohydrate. Kidney C A W shows an amino acid composition quite distinct from other CAs. Studies with purified CAIV show that this enzyme can form ionic channels in a lipid bilayer and thereby most efficiently translocate hydrogen or bicarbonate ions through the membrane [ 141. When the full mRNA sequence is available it will be interesting to see if CAIV shows both a signal sequence and the 'stop transfer' sequence which is thought to be essential t o the membrane integration function [ 15 I. G e m striit'riire litid regiil&)tr

Four of the CA genes. CAI, CAII, CAlIl and CAVll 18, 16-20], have been fully characterized. In all cases. the protein coding sequence is divided into seven exons and the locations of the intervening sequences are conherved. The mouse CAI1 gene shows a minor variation o f this pattern with intron IV displaccd 14 bp upstream from the position found in human CAI1 and all other CA genes. A more significant structural difference, shown by the mouse and human CAI genes is an additional large intron interrupting the 5' untranslated regions. The overall size of the CAI gene is, thereby, 3-5 times largcr than other CA genes, which vary from 9.8 kb for human CAVll t o 17 kb for chicken and human CAII. Dctailed plans of the architecture o f the CA genes provide the necessary tools with which t o investigate the mechanisms which regulate their expression, and what follows is a summary of current progress in this area. CAI

This cytoplasmic isoform is expresscd primarily in erythrocytes, but is found at lower levels in some other tissues including gastrointestinal epithelia. In erythroid ce!ls, the human CAI gene is activated late in fetal development at about the time of the switch from fetal to adult globin expression. Studies of adult erythroid differentiation using the inducible, mouse erytholeukaemia ( M E L ) cell system show that CAI transcription is highest in the uninduced state and decreases on induction, suggesting that CAI may be characteristic of the earliest stages of adult erythrogenesis [ 21 I.

~

~

~

~

N

R

I73

ISOENZY MES +1

-1

+1

CAI 29 000 260aa

NH,

COOH

I1 1 I I I I I 1 I I 1 I I I I I I I ! I I I I 1 +2+1

L I I J If

+2 -1-3+2

D IIfl

+2

B

+1

z

-3

1

rl

CAII, CAlll 29 000 259aa +45

u -

.~

[--:.:I I:

CAVl 36000 m307aa CAV 29 000 m c.260aa

Fig. 2. 7 1 1 ~.strirc~irrrqf' the sec.reterl ( ' A Vl protein coinpurtd with thut of' thtJ cytoplrrsinic ( ' A s ( 1 1 , ('All (ind C'Alll A signal sequence of between 16 and 26 residues is indicated at the N-terminus of CAVl by a broken box and the 4S-residue, hydrophilic. C-terminal extension [ 131 by a hatched box. T h e structure of the mitochondria1 CAV is not known. but many mitochondrial proteins are synthesized with transient, basic N-terminal leader peptides. indicated by broken box; data from 1 I21 suggests this may be cleaved between residues 2 I and 22. CAII and CAlll are shown divided into I0 amino acid segments. Hatched and filled boxes indicate additional and missing residues. respectively. in C A I and CAVl in comparison with CAI1 and C A M (For a review of membrancassociated protein structure see [ 4 I 1.1 Abbreviation: aa, amino acid.

Sequence analysis of CAI m R N A from erythrocytes indicates an intervening sequence in the 5' untranslated region, not found in other C A genes. T h e extra, upstream exon ( 6 9 bp in man; 62 b p in mouse) lies about 37 kb from the first coding exon in the human gene and between 1 0 and 2.50 kb in the mouse gene [ 19, 201. This 5' exon does not occur in mRNA transcribed in colon and is spliced to an acceptor in the 5' untranslated region of exon 1 of the colon mRNA [201. It appears that the erythrocyte and colon mRNAs are transcribed from two different promoters, one of which may be used only in erythrocytes and the other more ubiquitously. However, the colon promoter is not typical of a 'housekeeping. gene; there is no CpG-rich island and no examples o f CCGCCC S p 1-binding element>. Various questions arise from these studies. How is it possible to correctly transcribe the erythrocyte m R N A when the promoter is such a large distance upstream from the rest o f the gene'? Do the differences between the 5' non-coding regions of the erythrocyte and colon mRNAs confer differences in m R N A stability or translatability? Is the erythroid promoter more efficient than the colon promoter'? Sequence motifs characteristic of erythroid expression and found in the promoter regions o f globin genes have been identified in the CAI erythroid promoter. For example. thcre is a CCACACCC element at - 2 12 b p in the mouse C A I gene [20] and in the human gene there are five copies o f the 'signature' sequence GAATAG [ 221. Butterworth et ( J / . have recently shown that a GATAAG element at - 190 b p in the human C A I gene acts in cis by binding rruns-acting factors which occur in abundance only in erythroid cells 1221.

In contrast to the CAI promoters, the CAI1 promoter region appears t o be typical of a housekeeping gene [ 25 I; it displays a high G + C content, high C p G / G p C = 0.94. and nine Spl-binding sites. There is also a tandemly repeated -TCACCTCC- clement at - I 2 1 b p which seems t o fuhction as a 'housekeeping' element in the CAI1 gene. but also occurs in the promoter region o f /3-globin, human CAlll and certain other genes. Shapiro ct ( I / . 125I have explored the transcriptional activity of the human CAI1 promoter sequences by 5' deletion analysis in transfected HeLa and mouse L cells. These experiments identify species-specific differences in regulatory control such that. while the - 121 bp tandem repeats are required for optimal transcription in L cells. in the human cells transcription is more greatly influenced by the presence o r absence o f an Sp I-binding site. I S b p further upstream. T h e same authors have also investigated cell-type specific expression o f CAI1 and have focused on osteoclasts, because of the association between osteopetrosis and CAI1 deficiency. Treatment of the promyelocytic cell line HMO, which provides a model system for the study of osteoclastspecific exprcssion with the hormone 1,25-dihydroxyvitamin D, [ 1,25-(OH)2D,I.results in an induction de novo of both CAII protein ( 1 0-fold) and mRNA (20-fold) [ 26 1, This hormone is known t o affect strongly bone resorption and has been shown t o induce hypercalcaemia in chick embryos by stimulation of C A 1271. T h e 1,25(0H),D,-rcsponsive element in the CAI1 gene has yet to be identified and preliminary transfection experiments using the HL60 cells indicate that it is not in the immediate vicinity of the minimal promoter required for CAI1 expression in HcLa and mouse L cells.

('A11

T h e high activity isoform CAI1 is abundant in erythrocytes, but is also widely distributed in secretory and absorbing epithelia and is found in virtually all tissues. T h e clinical outcome of CAI1 deficiency is diverse and the human syndrome is characterized by osteopetro:is, renal tubular acidosis and cerebral calcification 1231. T h e picture is apparently not so severe in CAIl-deficient mice, but includes renal tubular acidosis and vascular calcification [24]. Vol. 18

C'AIII In man and most large mammals this isoform is found predominantly in red skeletal muscle with trace amounts in erythrocytes and smooth muscle [ 11. In rodents. CAM is also moderately abundant in liver and is largely confined to the perivenous hepatocytcs [27a]. Rats show a sexual dimorphism with C A M levels 10-20-fold higher in male than in female liver I28 I.

174

BIOCHEMICAL SOCIETY TRANSACTIONS

Low levels of C A M are detected in early fetal muscle; for with the C A M promoter and influences its expression in example, about 1% of adult levels at 10 weeks gestation in proliferating myoblasts. man. Substantial increases in both CAI11 protein and mRNA occur late in development in association with the differentiaI . Tashian. R. E. & Hcwctt-Emmett. I). (eds), ( 1984) N. Y. Auid. tion of adult muscle fibre types [29]. Histological analyses Sci. 429, 1-840 using antibodies and cDNA probes have established that 2. Tashian,R.E.(1989) HioEssciys 10, 186-102 CAlll is largely confined to type I , slow-twitch fibres 3. Davis, M. B., West. L. F.. Barlow. J. H.. Butterworth. P. H., 130, 311. In the course of fetal and infant growth, it appears Lloyd, J. C. & Edwards, Y. H. ( 1987) Somiit. Cell. M d . Genet. that the CAlll gene activity is modulated by suppression in 13, 173-178 4. Nakai, H., Byers, M. G., Venta. P. J., Tashian, R. E. & Shows, type 2, fast-twitch fibres and activation in type 1 fibres. The T. B. ( 1987) C‘yroh.etlet. C ’ l ~ I IG V I I V 44, ~ .234-235 suppression o f transcription in type 2 fibres appears to be 5 . Eicher, E. M., Stern, R. H.. Womack, J. E., Davisson, M. T., reversible and responsive to changing external influences. A Roderick, T. H. & Reynolds. S. C. ( 1976) Hiochem. Giwet. 14, generalized fast-to-slow-fibre-type transformation can be 6 5 1-660 induced by artifically altering the contractile activity of fast 6. Beechey, C., Twcedic. S., Spurr. N.. Ball. S.. Peters, J. & muscle using electrostimulators or by increased levels of Edwards, Y. H. ( I 990) Genomits in the press thyroid hormone 131, 321. This transformation is associated 7. Sutherland. G . R., Baker. E.. Rrnandez. K. E.. Callen. I>. F., with the induction of CAI11 mRNA and protein to slowAldred, P., Coghlan, J.. Wright. R. 61 Fernley, R. (1990) ($ogenet. Cell Genet. in the press twitch-fibre levels. Similar increases in CAlll are also 8. Montgomery, J. C. (1988) Ph.D. Thesis, University of Michiobserved after resection of the nerve supply to fast muscles gan. U.S.A. 1331. 9. Forsman, C., Behravan. G.. Jonsson, B., Liang. Z., Lindskog. S.. It seems likely that natural fluctuation in pituitary growth Ren, X., Sandstrom. J. & Wallgren, K. (1988) FEBS Letts. 228, hormone ( G H ) levels accounts for the sexual differences in 360-362 CAW expression in rat liver, such that hepatic C A M is 10. Tu, C., Paranawithana. S., Tanhauser, S., Jewell, D., Wynns. G., lowered in females by the continuous release of GH, but is Laipis, P. & Silverman. D. ( 1990) in the press not suppressed in males where G H is released in a pulsatory 1 1 . Eriksson, A. E. (1988) P h D . Thesis, University of Uppsala, fashion. This view finds support from studies in which G H Sweden levels have been modified artificially in both sexes 1341. It is 12. Hewett-Emmett. D., Cook, K. G. & Dodgson, S. J. (1988) Fed. I’roc. Fed. Am. Soc. Exp. B i d . 45, 166 1 noteworthy that levels of C A M are the same in male and female rat muscle and G H has no apparent influence on 13. Fernley, R. T. ( 1988) Trends Hiochem. S c i . 13, 356-359 muscle CAI11 expression. The possibility that the muscle and 14. Wistrand, P. J. ( 1984)Ann. N. Y. Auid. Sci. 429, 195-206 15. Yost, C., Hedgpeth, J.& Lingappa, V. ( 1983) Cell (C’cirnhridge, liver mRNAs are transcribed from separate promoters is Mass.) 34, 759-766 being explored, but initial RNAse protection assays suggest 16. Venta. P. J., Montgomery. J. C., Hewett-Emmett, D.. Wiebauer. that the two CAI11 mRNAs are identical at their 5‘ ends [ 3 5 ] . K. & Tashian, R. E. ( 1 985) J. Hiol. (’hem. 260, I 2 130- I 2 135 The promoter region associated with transcription of 17. Yoshihara, C. M., Lee, J. D. & Dodgson, J. B. ( 1987) Niccleic human muscle CAI11 is not characteristic of a ti Acids HCS. 15,753-770 18. Lloyd, J., Brownson. C.. Tweedie, S., Charlton. J. & Edwards, gene. Downstream of the TATA box there Y. H. ( 1987) Genes f k v . I , 594-602 sequence similar to a consensus element found in a number of housekeeping genes and CAII, there is moderate G + C 19. Brady, H., Lowe, N., Sowden, J.. Barlow, J. & Butterworth. P. H. 1988) Riochem. Soc. Trrms. 17, 184- I 85 content, a CpG/GpC ratio and two Spl-binding sites. These 20. (Fraser, P., Cummings, P. & Curtis, P. ( 1989) Mol. Cell Hiol. 9, sequences are methylation free in all tissues regardless of 3308-33 I3 expression, indicating the presence of a bona fide ‘HTF’ 2 1. Fraser, P. J. & Curtis, P. J. ( 1987) Genes flev. I , 855-886 island 1361. 22. Butterworth, P., Barlow, J., Brady, H.. Edwards, M., Lowe, N. & There is evidence of an upstream enhancer region about Sowden, J. ( 1990) in The Ciirhonic Anhydrases: CPllular Physiology and Molecular Genetics (Dodgson, S., ed.), Plenum 1.5 kb 5’ to the coding sequence. This 280 bp region conPress, New York. in the press tains several copies of motifs identified as transcriptionally important in the simian virus 40 early enhancer [37]. While 23. Sly, W. S., White, M. P., Sundaram, V., Tashian, R. E., HewettEmmett, D., Gurband, P., Vainsel, M., Baluarte, H., Grushkin, this enhancer allows ubiquitous expression, elements from A,, Al-Mosawi, A,, Sakati. N. & Ohlsson, A. ( 1 9 8 5 ) N. Engl. J . this region have been found in the immediate vicinity of Med. 313, 139-145 tissue-specific enhancers and, in particular, near to the 24. Lewis, S. E., Erickson. R. P., Barnett. L. B., Venta, P. J. & muscle-specific creatine kinase [ 381. Several other candidate Tashian, R. E. ( 1988) I’roc. Ncirl. Atnd. Sci. U.S.A. 85, regulatory sequences including possible thyroid hormone 1962-1966 receptor- and glucocorticoid receptor-binding elements have 25. Shapiro, L., Venta, P. & Tashian. R. E. ( 1987) Mol. Cell Biol. 7, 4589-4593 also been identified in the CAI11 upstream region and in 26. Shapiro, L., Venta, P.. Ya-Shiou. L. & Tashian, R. E. (I989) vifro gene expression systems are being used to establish FEBS Lett. 249,307-3 1 0 which of these candidate regulatory sequences are transcrip27. Narbaitz, R., Kacew, S. & Sitwell. L. (1984) Ann. N. Y. Accid. tionally active. Sci. 429,479-480 Preliminary transfection studies, in which the human 27a.Carter, N., Wistrand, P. J. & Lonnerholm, G. (1989) Acta CAlll promoter activity was compared in mouse C2C12 I’hysiol. Scand. in the press myoblasts and 1OT1/2 fibroblasts, indicate that the sequences 28. Carter, N. D., Jeffery, S. & Shiels, A. (1982) FERS Lett. 139, necessary for muscle-specific expression are contained 265-266 within 2.5 kb of sequence immediately 5’ to the coding 29. Jeffery, S., Edwards, Y. & Carter. N. ( 1980) Biochem. Genet. 18,843-849 sequence (S. Tweedie & Y. H. Edwards, unpublished work). A recent interesting observation is that the myogenic cell line 30. Wistrand, P., Carter, N. & Askmark, H. ( 1987) Comp. Biochem. Physiol. 86A. 171-284 23A2, derived from 10T1/2 cells after treatment with 531. Brownson, C., Isenberg, H., Brown, W., Salmons, S. & azacytidine, expresses CAI11 at higher levels than found in Edwards, Y. ( 1 988) Muscle Nerve 1 1, 1 183- 1 189 other myoblast lines and in early fetal muscle. Both C2C12 32. Fremont, P.,Lazure, C.. Tremblay. R., Chretien, M. & Rogers, and 23A2 cells are known to express the myogenic determiP. A. ( 1 987) Biochem. Cell Biol. 65.790-799 nant MyoD1, a phosphoprotein which probably acts as a 33. Carter, N., Wistrand, P., Isenberg. H.. Askmark, H., Jeffery, S., transcription factor in early myogenesis [39,40]. It will be of Hopkinson, D. & Edwards, Y. (1988) Biochern. J. 256, 147-1 52 some interest to determine whether this protein interacts 1990

ISOENZY MES

175

34. Jeffery. S.. Wilson. C.. Mode. A,, Gustal’sson. J.-A. 8 Carter. N. ( 1986) J. Errtloc~rirroi.110. 113- I 2 6 35. Edwards, Y. H. ( 1990) in 7%o Citrhoriic Ari/rylrct.srs: C’c.lhtlerr /’/tyithgx trtrd hkdcwt/ttr (;c~tic~iic:s(Ihdgson. S.. ed.), Plenum Press, New York. in the press 36. Edwards, Y.. Charlton. J. 8 Hrownson. C. (1988) C;cwe 71, 173-18 I 37. Zeuke. M.. Grundstrom. ‘I., Matthes. H., Wintzerith. M.. Schatz. C.. Wildernon, A. Ji Chambon. P. ( 10861 Ii.Z/HO .I. 8, 62-70

38. Jaynes. J.. Johnson, J.. Buskin. J.. Gartside, C. 8 Hauscka. S. ( 1988) Mol. (’ell. Rioi. 8. 62-70 39. Tweedie, S. & Edwards. Y. ( 1989) Nioc,/rerri. (;cric,/. 27, 17-30 4).Kelly. C., Carter, N., Jeffery, S. Ji Edwards, Y. ( I YXX) Hiosci. KC/>.8, 40 1-106 41. Wickner. W. T. & Lodisa. H. F. ( 1985) .Sc,icwc,e, 230, 400-408

Keccivrd I0 October I 989

Glutathione S-transferases TIMOTHY J. MANTLE. FIONA M. MKUSKER. MICHAEL PHILLIPS and SINEAD BOYCE Departi?icvit c?f’Hioc~li~~rrri.str?: Iritiitjt C b l k g ~Dirh ~ fill 2 Nepiihlic cf’lri~l(itid Striictiirc~t i r i d iic~i~iCii(’I~itiirfJ.s

The cytosolic glutathione Stransferases (GSTs) are homoand hetero-dimers made up of subunits (A!, approx. 25 000) that can be grouped into three gene families; alpha, mu and pi 1 I ] . In the rat. the most-studied species, full-length cDNA sequences have been described for several subunits. For most o f these sequences, the corresponding protein subunit has been identified. although it should be noted that only in the case of Yb, is the complete amino acid sequence available 121. This is not a trivial point, as it is clear that considerable microheterogeneity exists ( 3I and only when purified forms have been completely sequenced can identity with cDNA sequences be established. Quaternary structure appears t o follow the rule that homo- and hetero-dimers can be formed from subunits within one of the three gene families, but not between families, and is best represented by the shorthand notation Yb,Yb,, etc. The alternative nomenclature which describes the various subunits by an arabic numeral 141 is not capable o f identifying thc various subunits as being members of one o f the three gene families and suffers accordingly. It is clear that we are still at a ‘stamp-collecting‘ phase in describing the multiple forms. so that any further rationalizations of nomenclature at the present time would have to be half-way-house measures. It is clear, however. that three welldocumented gene families exist and that if, for example, the Yc and Y k subunits were renamed Ya, and Ya,, then Ya, Yb and Yf families could conveniently be described and would correspond to classes alpha, mu and pi, respectively. Clearly the Yn-subunit 151. which is now known to be heterogeneous [6], would have to be renamed as Yb type and indeed the Yb, cDNA reported by Abramowitz & Listowsky [7] is clearly Yn, 161. 7issire-specificexpression There is a marked tissue-specific expression o f particular subunits which has been documented at the rotein level [8-10] and also at the level o f mRNA 17, 1 1 - 1 3y This specificity of expression has been revealed to be even more complicated at the histological level (114, 151; S. Boyce & T. J. Mantle, unpublished work). As a general rule, most epithelial cells express high levels of several subunits, and in the case of hepatocytes, kidney tubular epithelia and the epithelia of the gastrointestinal tract, the particular complement of GST subunits represents a significant fraction of the total cytosolic protein. Considerable work on the regulation of expression Abbreviations used: GST. glutathione .S-transferase; BP, binding protein.

Vol. 18

of the rat Ya gene has been rcported by Pickett and coworkers (for a review see 131) and several 5’ regulatory sequences have been identified. I’roi~~~s~~dfiriit~tioirtrl roles. Early work by Arias ci ril. I 161. Litwack et al. [ 171 and Jakoby & Habig 1181 suggested that the high level of expression o f GSTs reflected two distinct functional roles played by these proteins. The first is that of an intracellular albumin providing a thermodynamic sink in the cytosol for compounds with diverse structures, but a common feature of hydrophobicity (see, for example 1191). The functional relationship between the binding properties of the GSTs and the growing family of low-M, binding proteins (BPs), e.g. fatty acid BP and retinoic acid BP needs to be explored. There is often ccmsiderable traffic o f hydrophobic ligands across epithelial cells, f o r example. approximately 2 0 g of bile acids cross hepatocytes from the sinusoidal face t o the bile canalicular face each day 1201. The second function o f members o f this super-gene family is clearly a protective role against potentially toxic elcctrophiles. As their name denotes, these enzymes catalyse a conjugation reaction with glutathione if the hydrophobic ligand contains an electrophilic centre, which may be carbon, oxygen, nitrogen or sulphur 12 11. There has been discussion as to the evolution o f the substrate specificitics o f thc multiple forms, particularly as the most efficient substrates i r i vitro, e.g. I-chloro-2.4-dinitrobenzene (CDNB), are unlikely t o be encountered iri t i w . Recently. several products of oxidative stress, e.g. 4-hydroxyalkenals [22. 231, thymine and DNA hydroperoxides 1241. and lipid hydroperoxides 175, 261, have been shown to be substrates for various GSTs providing evidence for the hypothesis that these enzymes may play a role in neutralizing the pathophysiological products of oxidative metabolism 1271. However, it should not be forgotten that plants and micro-organisms produce a wide range o f structurally diverse end-products o f metabolism, many o f which contain electrophilic centres or which can be metabolized by the cytochrome 1’-450 system to produce electrophilic species (e.g. aflatoxin). Similar comment can be made about environmental/industrial pollutants, e.g. polycyclic hydrocarbons. For further details on the production of benz( u)pyrene diol epoxides, and their subsequent conjugation reactions with glutathione, sce, for example 128,29]. Kinetic stirdies

In most cases the k,,, / K t , ,values arc not high and this may be compensated for by the high concentration of several o f these enzymes, particularly in liver. Mannervik and coworkers have shown that, at least for the Yb,Yb, and YaYc heterodimers, the subunits are kinetically independent 1301. Early kinetic studies with Yb,Yb, were interpreted in favour of a Ping-Pong mechanism at low concentrations of glutathione which switched to a sequential mechanism at high concentrations [3I ] . However, a later analysis o f the original data set has been suggested to be more consistent with a steady-state random mechanism 132) Recent studies with the