TiPS - March 1992 [Vol. 131
102 possibly be formed by the rea&on of white lead with lime: PbCo3 + Ca(OH)z+ Pb(OH)z+ CaC03 ......-.. (7) This is probably a Sk@% cation, as lead is an amphoteric element, and also forms plumbates such as Pb(OH)a’-. However, all the forms of basic lead oxide can be expected to react with sulfide to yield black lead sulfide. Where would the sulfide originate? Sulfide is given off by decomposing bodies, accounting for the frescoes closest to the tombs being most affected. Sulfide is also exhaled and transpired in low amounts%om live humans, deriving from the sulfane pool. Sulfane sulfur is a sulfur bound to another sulfur. Sulfanes include thiosuIfate, persulfides and thiosulfonates. Sulfane sulfur derives from 3-mercaptopyruvate via the enzyme mercaptopyruvate sulfurtransferase (EC 2.8.1.2). Mercaptopyruvate, in turn, is formed from cysteine by means of the enzyme cysteine aminotransferase (EC 26.1.3). Mercaptopyruvate sulfurtransferase also catalyses the reaction of thiols with persulfides, yielding hydrogen sulfide:
dation to colored lead oxides, such as Pb304: 3PbC03 + 3Ca(OH)z + l/202 + Pb304 + 3CaC03 + 3H20 ........ (9) Other artists seem quickly to have learned from Cimabue’s error, for I know of no other frescoes in which white lead was used. Acknowledgements I thank Klaus Brendel for help with the chemistry of lead. B. MAX
References 1 Eggleton, P. and Eggleton, G. P. (192n Biochem. 1. 21,19O-195 2 Egg&on, P. &td Eggleton, G. P. (1927) J. Physiol. ILondJ63,15~161 3 Fiske, C. H. and Subbarow, Y. (1927) Science 65,401-4O3 4 Fiske, C. H. and Subbarow, Y. (1929) J. Biol. Chem. 81. 62-79 5 Lundsgaard, E. (1930) Biochem. Z. 217, 162-177 6 Needham, D. M. (1960) in The Strutture and Function of Muscle (Boume, G. H., ed.), pp. 55-104, Academic Press 7 Mommaerts, W. F. H. M. (1950) Mwscular Contruction: A Topic in Molecular Physiology, Interscience Publishers 8 Lipmann, F. (1941) Ado. Enzymol. 1, 99-162 9 Wilkie, D. (1981) Cibn Found. Symp. 82, 102-119
10 Harris, R. C., Hultman, E. and Nordesiii, L-O. (1974) Stand. 1. Clin. Lab. In;est. 33, 109-120 11 Atkinson, D. E. (1968) Biochemistry 7, 4030-4034 12 Ennor. A. H. and Morrison. 1. F. (195g)‘Physiol. Rev. 38, 631-674 ’ _ 13 Thoai. N. V. and Robin, Y. (19541 Biochim. Biophys. Actn 13,533-536 14 Makisumi, S. (1961) J. Biochem. (Tokyo) 49,2&I--291 15 Beigquist, P. R. and Hartman, W. D. (1969) Mar. Biol. 3,247-268 16 Allen, J. A. and Garrett, M. R. (1971) Adv. Mar. Biof. 9,205-253 17 Surholt, B. (1979) Eur. /. Biochem. 93, 279-300 18 Kassab, R., Pradel, L-A. and Thoai, N. V. (1965) Biochim. Biophys. Acta 99, 397-405 19 Shields, R. P. and Whitehair, C. K. (1973) &I. J. Biochem. 51,104&1049 20 Fitch, C. D., Jellinek, M. and Mueller, E. J. (1974) J. Biol. Chem. 249, 106&1063 21 Ohira, Y., Ishine, S., Inoue, N. and Yunoki, K. (1991) Biochim. Biophys. Actu Mol. Basis Dis. 1097, 117-122 22 Mudd, S. H. and Poole, J. R. (1975) Metabolism 24, 721-735 23 Brown, D.. A. (1983) Leonardo’s Last Supper: The Restoration, National Gallery of Art, Washington 24 Feibusch, H. (1946) MuraZ Painfiug, Adam and Charles Black 25 Hollernan, A. F. (1960) Lehrbuch der Anorganischen Chemie, Walter de Gruyter & Co. 26 Nicholson, A. (1932) Cimabue - A Critical Study, Kennikat Press 27 Chiellini, M. (1988) Cimabue, Scala Books
R!%H+ RsH+RSSR + H,S .._........ (8) The enzyme is found in all tissues, being especially high in liver and kidney. As a result, wherever there are humans, there are low levels of sulfide. A little black goes a long way on a white wail, so not much is needed to darken a fresco. In one experiment, I mixed gleaming white suspensions of lead carbonate and calcium hydroxide together, and added a few drops of sodium sulfide solution. Immediately, the suspension turned grey-black. However, even without the addition of sulfide, the stirred suspension changed color; in this case to a pale yellow. This must be caused by precipitation of a small amount of lead oxide, or litharge. Indeed, Cimabue’s Assisi frescoes have an overall yellowish tone. Even without the presence of sulfide, therefore, white lead, or lead carbonate, is an unsuitable pigment for use in fresco. In darkened oil paintings, lead sulfide can be oxidized to white lead sulfate with dilute hydrogen peroxide. This is not effective in fresco, however, as the basicity leads to oxi-
Doubt expressed about identity of remaining orphan clone
RDCl may not be VIP receptor The TiPS Receptor Nomenclature Supplement 1992 reports that the human VIP receptor has been cloned. However, we have reason to doubt that this is the correct interpretation for the protein quoted (GenBank accession number ~64749). In 1989, some of us2 reported the cloning of four ‘orphan’ G protein-coupled receptors from a dog thyroid cDNA library. Three of these, RDC4, RDC7 and RDC8, have since been identified as 5-HT10, adenosine Al and adenosine A2 receptors, respectivelF. Sreedharan et al. reported the human homolog of the fourth receptor, RDCl, to be the VIP receptor’. However, neither in @ 1992,l!lsevler Science Publishers Ltd (UK)
Japan nor in Belgium have we been able to confirm this identity by ‘251-labelled VIP binding to membranes of COS cells transfected with RDCl or its rat homolog, or by stimulation by VIP of adenylyl cyclase activity. Some of us (S.N. and T.I.) have, however, found low levels of 1251-labelled VIP binding to untransfected cells, which might explain the binding data published by Sreedharan et al. Lack of homology of RDCl with the recently cloned secretin receptoI$ also throws doubt on the identity of this clone. Homology might be predicted since secretin and VIP are very closely related peptides, and high concentrations
TiPS - March 1992 [Vol. 131
103
of VIP can interact with, and activate, the cloned secretin receptor expressed in COS cells. SHIGEKAZU NAGATA, ISHIHARA,
TAKESHI
PATRICK ROBBERECHT+,
FREDERICK LIBERT+, MARC PARMENTIER’,
JEAN CHRISTOPHE*
AND GILBERT VA§SART+
Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, 565 Osaka, Japan. ‘Laboratoire de Chemie Biologique and Instituf de Reckercke lnterdisciplinaire and ‘Faculty of Medicine, Free University of Brussels, 808 route .de Lennik, 2070 Brussels, Belgium.
References 1 Sreedharan, Peterson,
S. P., Robichon, A., K. E. and Goetzl, E. J. (1991)
Molecular characterization of endothelin receptors Takeshi Sakurai, Masashi Yanagisawa and Tomoh Masaki Following the first report on the identification of endothelin (ET), an increasing body of work has accumulated on this endothelium-derived 22-amino acid vasoconstictor peptide. Subsequently, the existence of three distinct isoforms of ET, designated ET-I, ET-2 and ET-3, was predicted from the finding of three separate genes. The differential potencies of the three isoforms of the ET family have opened up the possibility of the existence of multiple ET receptor subtypes. Recently, molecular biological techniques provided direct evidence of at least two distinct subtypes of ET receptor. This article discusses the functions of the ETs, focusing especially on the molecular characteristics of their receptors. Because of their unique structural and pharmacological properties, the endothelins (ETs) are attracting the attention and interest of many investigators in all fields of biomedical research. ET-l is the most potent vasopressor peptide yet characterized and its action is extremely long lasting, such that when it is administered as an intravenous bolus injection, a prolonged pressor response is observed, lasting several hours’. There are now many indications that the endothelins are involved in regulation of the vascular system as well as in the function of
T. Sakurai is a Research Scientist in the Department of Pharmacology, Institute of Basic Medical Sciences, Universityof Tsukuba, Tsukuba, Ibaraki, 305 Japan, M. Yanagisawa is Associate Professor at the Howard Hughes Medical Institute and Departmenf of Molecular Genetics, University of Texas South Western Medical Center, Dallas, TX 75235, USA, and T. Masaki is Professor of Pkarmacology, Faculty of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606 Japan.
several other systems, and there is also evidence suggesting that the ETs may be involved in the pathophysiological mechanism of a number of vascular conditions, such as hypertension2, acute renal failure3, angina pectoris* and other related pathological conditions. ET-l is processed from an -200 amino acid residue precursor (preproET-1) through the formation of a 38- or 39-residue (depending on the species) intermediate form called ‘big ET-l’. An unusual proteolytic cleavage of big ET-l between the Trp21 and Va122 residues performed by an ‘endothelin-converting enzyme’ generates mature ET-l, as well as a C-terminal fragment. Although big ET-1 is only 1% as potent as ET-1 in inducing contractile activity in vascular strips, it is equally potent in raising blood pressure in vivo, since big ET-1 is rapidly converted to active ET-l by endothelinconverting enzyme. This enzyme is believed to be a novel neutral met-
alloendopeptidase, and is efficiently
Proc. Nat! Acad. Sci. USA 88.4986-4990 2 Libert, F. et al. (1989) Science 244,569-572 3 Maenhaut, C. et al. (1991) Biockem. Biopkys. Res. Commun. 173.1169-1178 4 Lib&, F. et al. (1991) EMBO J. 10, 1677-1632 5 Maenhaut, C. et al. Biockem. Biopkys. Res. Commun. (in press) 6 Ishihara, T. et al. (1991) EMBO J. 10, 1635-1641
inhibited by the metalloprotease inhibitor phosphoramidons. The existence of three distinct genes that potentially encode three isopeptides of the ET family, ET-l, ET-2 and ET-3, was subsequently demonstrated in human and other mammalian genomes6. ET-2 and ET-3 differ from ET-l in two and six of their amino acid positions, respectively. All three of the family members possess two intra-chain disulfide bridges, a feature shared by the sequence of sarafotoxins (found in Me venom of an Israeli burrowing asp), suggesting that the ETs and sarafotoxins share a long genealogical history7 (Fig. 1). The three ET family members are expressed in various tissues with different distribution patterns; only ET-l is detected in vascular endothelial cells. ET-2 and ET-3 are expressed in other tissues, such as the brain, kidney, adrenal gland and intestines”. ET-l is also expressed in various non-vascular cells within the brain, kidney, lung and other tissues”. Apart from their potent and extremely long-lasting vasoconstrictor activities, the ETs elicit a wide spectrum of both vascular and non-vascular actions in a variety of tissues, including airways (bronchoconstriction), kidney (various glomerular, tubular and endocrinological effects), myocardium (positive inotropic and chronotropic actions), and central and peripheral nervous system (modulation of neurotransmission and release of neuroendocrine hormones)“. Heterogeneity of receptors These diverse responses induced by ETs are mediated by specific ET receptors. Numerous studies suggest that the pharmacological responses induced by ET peptides may not be mediated by a single type of receptor. When ETs are injected intravenously into animals, they elicit Q 192,flswier Science Publishers Ltd(UK)
102 possibly be formed by the rea&on of white lead with lime: PbCo3 + Ca(OH)z+ Pb(OH)z+ CaC03 ......-.. (7) This is probably a Sk@% cation, as lead is an amphoteric element, and also forms plumbates such as Pb(OH)a’-. However, all the forms of basic lead oxide can be expected to react with sulfide to yield black lead sulfide. Where would the sulfide originate? Sulfide is given off by decomposing bodies, accounting for the frescoes closest to the tombs being most affected. Sulfide is also exhaled and transpired in low amounts%om live humans, deriving from the sulfane pool. Sulfane sulfur is a sulfur bound to another sulfur. Sulfanes include thiosuIfate, persulfides and thiosulfonates. Sulfane sulfur derives from 3-mercaptopyruvate via the enzyme mercaptopyruvate sulfurtransferase (EC 2.8.1.2). Mercaptopyruvate, in turn, is formed from cysteine by means of the enzyme cysteine aminotransferase (EC 26.1.3). Mercaptopyruvate sulfurtransferase also catalyses the reaction of thiols with persulfides, yielding hydrogen sulfide:
dation to colored lead oxides, such as Pb304: 3PbC03 + 3Ca(OH)z + l/202 + Pb304 + 3CaC03 + 3H20 ........ (9) Other artists seem quickly to have learned from Cimabue’s error, for I know of no other frescoes in which white lead was used. Acknowledgements I thank Klaus Brendel for help with the chemistry of lead. B. MAX
References 1 Eggleton, P. and Eggleton, G. P. (192n Biochem. 1. 21,19O-195 2 Egg&on, P. &td Eggleton, G. P. (1927) J. Physiol. ILondJ63,15~161 3 Fiske, C. H. and Subbarow, Y. (1927) Science 65,401-4O3 4 Fiske, C. H. and Subbarow, Y. (1929) J. Biol. Chem. 81. 62-79 5 Lundsgaard, E. (1930) Biochem. Z. 217, 162-177 6 Needham, D. M. (1960) in The Strutture and Function of Muscle (Boume, G. H., ed.), pp. 55-104, Academic Press 7 Mommaerts, W. F. H. M. (1950) Mwscular Contruction: A Topic in Molecular Physiology, Interscience Publishers 8 Lipmann, F. (1941) Ado. Enzymol. 1, 99-162 9 Wilkie, D. (1981) Cibn Found. Symp. 82, 102-119
10 Harris, R. C., Hultman, E. and Nordesiii, L-O. (1974) Stand. 1. Clin. Lab. In;est. 33, 109-120 11 Atkinson, D. E. (1968) Biochemistry 7, 4030-4034 12 Ennor. A. H. and Morrison. 1. F. (195g)‘Physiol. Rev. 38, 631-674 ’ _ 13 Thoai. N. V. and Robin, Y. (19541 Biochim. Biophys. Actn 13,533-536 14 Makisumi, S. (1961) J. Biochem. (Tokyo) 49,2&I--291 15 Beigquist, P. R. and Hartman, W. D. (1969) Mar. Biol. 3,247-268 16 Allen, J. A. and Garrett, M. R. (1971) Adv. Mar. Biof. 9,205-253 17 Surholt, B. (1979) Eur. /. Biochem. 93, 279-300 18 Kassab, R., Pradel, L-A. and Thoai, N. V. (1965) Biochim. Biophys. Acta 99, 397-405 19 Shields, R. P. and Whitehair, C. K. (1973) &I. J. Biochem. 51,104&1049 20 Fitch, C. D., Jellinek, M. and Mueller, E. J. (1974) J. Biol. Chem. 249, 106&1063 21 Ohira, Y., Ishine, S., Inoue, N. and Yunoki, K. (1991) Biochim. Biophys. Actu Mol. Basis Dis. 1097, 117-122 22 Mudd, S. H. and Poole, J. R. (1975) Metabolism 24, 721-735 23 Brown, D.. A. (1983) Leonardo’s Last Supper: The Restoration, National Gallery of Art, Washington 24 Feibusch, H. (1946) MuraZ Painfiug, Adam and Charles Black 25 Hollernan, A. F. (1960) Lehrbuch der Anorganischen Chemie, Walter de Gruyter & Co. 26 Nicholson, A. (1932) Cimabue - A Critical Study, Kennikat Press 27 Chiellini, M. (1988) Cimabue, Scala Books
R!%H+ RsH+RSSR + H,S .._........ (8) The enzyme is found in all tissues, being especially high in liver and kidney. As a result, wherever there are humans, there are low levels of sulfide. A little black goes a long way on a white wail, so not much is needed to darken a fresco. In one experiment, I mixed gleaming white suspensions of lead carbonate and calcium hydroxide together, and added a few drops of sodium sulfide solution. Immediately, the suspension turned grey-black. However, even without the addition of sulfide, the stirred suspension changed color; in this case to a pale yellow. This must be caused by precipitation of a small amount of lead oxide, or litharge. Indeed, Cimabue’s Assisi frescoes have an overall yellowish tone. Even without the presence of sulfide, therefore, white lead, or lead carbonate, is an unsuitable pigment for use in fresco. In darkened oil paintings, lead sulfide can be oxidized to white lead sulfate with dilute hydrogen peroxide. This is not effective in fresco, however, as the basicity leads to oxi-
Doubt expressed about identity of remaining orphan clone
RDCl may not be VIP receptor The TiPS Receptor Nomenclature Supplement 1992 reports that the human VIP receptor has been cloned. However, we have reason to doubt that this is the correct interpretation for the protein quoted (GenBank accession number ~64749). In 1989, some of us2 reported the cloning of four ‘orphan’ G protein-coupled receptors from a dog thyroid cDNA library. Three of these, RDC4, RDC7 and RDC8, have since been identified as 5-HT10, adenosine Al and adenosine A2 receptors, respectivelF. Sreedharan et al. reported the human homolog of the fourth receptor, RDCl, to be the VIP receptor’. However, neither in @ 1992,l!lsevler Science Publishers Ltd (UK)
Japan nor in Belgium have we been able to confirm this identity by ‘251-labelled VIP binding to membranes of COS cells transfected with RDCl or its rat homolog, or by stimulation by VIP of adenylyl cyclase activity. Some of us (S.N. and T.I.) have, however, found low levels of 1251-labelled VIP binding to untransfected cells, which might explain the binding data published by Sreedharan et al. Lack of homology of RDCl with the recently cloned secretin receptoI$ also throws doubt on the identity of this clone. Homology might be predicted since secretin and VIP are very closely related peptides, and high concentrations
TiPS - March 1992 [Vol. 131
103
of VIP can interact with, and activate, the cloned secretin receptor expressed in COS cells. SHIGEKAZU NAGATA, ISHIHARA,
TAKESHI
PATRICK ROBBERECHT+,
FREDERICK LIBERT+, MARC PARMENTIER’,
JEAN CHRISTOPHE*
AND GILBERT VA§SART+
Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, 565 Osaka, Japan. ‘Laboratoire de Chemie Biologique and Instituf de Reckercke lnterdisciplinaire and ‘Faculty of Medicine, Free University of Brussels, 808 route .de Lennik, 2070 Brussels, Belgium.
References 1 Sreedharan, Peterson,
S. P., Robichon, A., K. E. and Goetzl, E. J. (1991)
Molecular characterization of endothelin receptors Takeshi Sakurai, Masashi Yanagisawa and Tomoh Masaki Following the first report on the identification of endothelin (ET), an increasing body of work has accumulated on this endothelium-derived 22-amino acid vasoconstictor peptide. Subsequently, the existence of three distinct isoforms of ET, designated ET-I, ET-2 and ET-3, was predicted from the finding of three separate genes. The differential potencies of the three isoforms of the ET family have opened up the possibility of the existence of multiple ET receptor subtypes. Recently, molecular biological techniques provided direct evidence of at least two distinct subtypes of ET receptor. This article discusses the functions of the ETs, focusing especially on the molecular characteristics of their receptors. Because of their unique structural and pharmacological properties, the endothelins (ETs) are attracting the attention and interest of many investigators in all fields of biomedical research. ET-l is the most potent vasopressor peptide yet characterized and its action is extremely long lasting, such that when it is administered as an intravenous bolus injection, a prolonged pressor response is observed, lasting several hours’. There are now many indications that the endothelins are involved in regulation of the vascular system as well as in the function of
T. Sakurai is a Research Scientist in the Department of Pharmacology, Institute of Basic Medical Sciences, Universityof Tsukuba, Tsukuba, Ibaraki, 305 Japan, M. Yanagisawa is Associate Professor at the Howard Hughes Medical Institute and Departmenf of Molecular Genetics, University of Texas South Western Medical Center, Dallas, TX 75235, USA, and T. Masaki is Professor of Pkarmacology, Faculty of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606 Japan.
several other systems, and there is also evidence suggesting that the ETs may be involved in the pathophysiological mechanism of a number of vascular conditions, such as hypertension2, acute renal failure3, angina pectoris* and other related pathological conditions. ET-l is processed from an -200 amino acid residue precursor (preproET-1) through the formation of a 38- or 39-residue (depending on the species) intermediate form called ‘big ET-l’. An unusual proteolytic cleavage of big ET-l between the Trp21 and Va122 residues performed by an ‘endothelin-converting enzyme’ generates mature ET-l, as well as a C-terminal fragment. Although big ET-1 is only 1% as potent as ET-1 in inducing contractile activity in vascular strips, it is equally potent in raising blood pressure in vivo, since big ET-1 is rapidly converted to active ET-l by endothelinconverting enzyme. This enzyme is believed to be a novel neutral met-
alloendopeptidase, and is efficiently
Proc. Nat! Acad. Sci. USA 88.4986-4990 2 Libert, F. et al. (1989) Science 244,569-572 3 Maenhaut, C. et al. (1991) Biockem. Biopkys. Res. Commun. 173.1169-1178 4 Lib&, F. et al. (1991) EMBO J. 10, 1677-1632 5 Maenhaut, C. et al. Biockem. Biopkys. Res. Commun. (in press) 6 Ishihara, T. et al. (1991) EMBO J. 10, 1635-1641
inhibited by the metalloprotease inhibitor phosphoramidons. The existence of three distinct genes that potentially encode three isopeptides of the ET family, ET-l, ET-2 and ET-3, was subsequently demonstrated in human and other mammalian genomes6. ET-2 and ET-3 differ from ET-l in two and six of their amino acid positions, respectively. All three of the family members possess two intra-chain disulfide bridges, a feature shared by the sequence of sarafotoxins (found in Me venom of an Israeli burrowing asp), suggesting that the ETs and sarafotoxins share a long genealogical history7 (Fig. 1). The three ET family members are expressed in various tissues with different distribution patterns; only ET-l is detected in vascular endothelial cells. ET-2 and ET-3 are expressed in other tissues, such as the brain, kidney, adrenal gland and intestines”. ET-l is also expressed in various non-vascular cells within the brain, kidney, lung and other tissues”. Apart from their potent and extremely long-lasting vasoconstrictor activities, the ETs elicit a wide spectrum of both vascular and non-vascular actions in a variety of tissues, including airways (bronchoconstriction), kidney (various glomerular, tubular and endocrinological effects), myocardium (positive inotropic and chronotropic actions), and central and peripheral nervous system (modulation of neurotransmission and release of neuroendocrine hormones)“. Heterogeneity of receptors These diverse responses induced by ETs are mediated by specific ET receptors. Numerous studies suggest that the pharmacological responses induced by ET peptides may not be mediated by a single type of receptor. When ETs are injected intravenously into animals, they elicit Q 192,flswier Science Publishers Ltd(UK)
