- Email: [email protected]
[61] Applications of amino acid analogs for studying co- and posttranslational modifications of proteins
[61]
AMINO
ACID ANALOGS
777
6). Furthermore, it was determined that the sizes of the cotranslationally modified lysosomal polypeptides were unaffected by the proteases unless detergent was added, in which case digestion proceeded to yield acidsoluble fragments, indicating that the polypeptide had been completely transferred across the membrane and segregated into the microsomal lumen, as is the case with secretory proteins. 22 22 G. Blobel and B. Dobberstein, J. Cell Biol. 67, 835 (1975).
[61] A p p l i c a t i o n s o f A m i n o A c i d A n a l o g s for S t u d y i n g Coand Posttranslational Modifications of Proteins B y G L E N H O R T I N a n d IRVING B O I M E
Synthesis of proteins often requires modification of the primary translation products, and numerous types of covalent modification of peptide chains have been observed. These changes can occur on the protein during its synthesis (cotranslational) or after release from the ribosome (posttranslational). Understanding the assembly of mature proteins requires definition of the mechanisms of these reactions and of the structural determinants that limit them to particular sites in the cellular environment. One approach to this question is to incorporate amino acid analogs into protein and to examine the consequent effects on processing. There are several examples of such applications of amino acid analogs. Baltimore and Jacobson ~used them to inhibit the proteolytic cleavage of viral polyproteins and thus establish that all the peptides were derived from a single precursor. Hydroxyproline synthesis in collagen was probed with proline analogs,2 clarifying the mechanism of this hydroxylation reaction. Proteolytic cleavage of prosomatostatin, proinsulin, 3 and pro-opiomelanocortin4,5 were inhibited by analogs of arginine and lysine. Asparagine-linked glycosylation was inhibited by analogs of threonine 6 and asparagine, 7 yielding insights into the specificity and mechanism of this reaction. Analogs of i M. F. Jacobson and D. Baltimore, Proc. Natl. Acad. Sci. U.S.A. 61, 77 (1968). 2 A. A. Gottlieb, Y. Fujita, S. Udenfriend, and B. Witkop, Biochemistry 4, 2507 (1965). 3 B. D. Noe, J. Biol. Chem. 256, 4940 (1981). 4 p. Crine and E. Lemieux, J. Biol. Chem. 257, 832 (1982). 5 H. Hoshina, G. Hortin, and I. Boime, Science 217, 63 (1982). 6 G. Hortin and I. Boime, J. Biol. Chem. 255, 8007 (1980). 7 G. Hortin, I. Boime, A. M. Stern, B. Miller, and R. H. Abeles, J. Biol. Chem. 257, 4047 (1983).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
778
TARGETING: SELECTED TECHNIQUES
[61]
leucine and threonine have been useful for probing the process by which secretory proteins traverse the membrane of the endoplasmic reticulum 8-~° and for examining the specificity of the protease that excises amino-terminal prepeptides from most secretory proteins.)1 The diversity of these examples illustrates the versatility of this approach. Virtually any process dependent on protein structure can be examined in this manner. Use of amino acid analogs has several characteristics that are often advantageous. 1. Proteins are modified cotranslationally, permitting analysis of modification reactions that occur on nascent peptide chains. 2. Subtle modifications of protein structure can be introduced because most analogs are structurally very similar to the amino acids they supplant. 3. Protein can be labeled with new heteroatoms such as selenium, chlorine, and fluorine. Fluorine, in particular, has been useful as a probe for nuclear magnetic resonance (NMR) spectroscopy of proteins. 12 4. They can be used in any system--intact organisms, organ culture, cell culture, and cell-free systems. Owing to the great variety of applications of this approach several general considerations are presented here rather than a detailed protocol with restricted applicability. Selection of Amino Acid Analogs A large number of amino acid analogs have been reported to be incorporated into protein. Some of these compounds are listed in the table. Analogs probably can be found to replace all the amino acids with the possible exception of glycine, alanine, and serine. A large number of potential analogs have not been studied thoroughly. Numerous additional amino acid antagonists are listed in other references. J3-j5 Some analogs listed in the table may be effective only in particular systems. For exams G.
Hortin and I. Boime, Proc. Natl. Acad. Sci. U.S.A. 77, 1356 (1980). 9 G. Hortin and I. Boime, J. Biol. Chem. 255, 7051 (1980). ~0p. Walter, I. Ibrahimi, and G. Blobel, J. Cell Biol. 91, 545 (1981). 11 G. Hortin and I. Boime, Cell 24, 453 (1981). 12 B. D. Sykes, H. I. Weingarten, and M. J. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 71, 469 (1974). 13 K. Dittmer, Ann. N . Y . Acad. Sci. 52, 1274 (1950). 14 W. Shive and C. G. Skinner, in "Metabolic Inhibitors" (R. M. Hochster and J. H. Quastel, eds.), p. 2. Academic Press, New York, 1963. ~5G. L. Igloi, F. yon der Haar, and F. Cramer, Biochemistry 17, 3459 (1978).
[61]
AMINO ACID ANALOGS
779
AMINO ACID ANALOGS INCORPORATED INTO PROTEIN
Amino acid Arginine Asparagine Aspartic acid Histidine
Isoleucine
Leucine Lysine
Analog Canavanine
threo-3-Fluoroasparagine threo-3-Fluoroaspartic acid erythro-3-Fluoroaspartic acid 2-Fluorohistidine 1,2,4-Triazole-3-alanine 2-Methylhistidine 3-Amino- 1,2,4-triazoylalanine 4-Thiaisoleucine O-Ethylthreonine O-Methylthreonine 4-Fluoroisoleucine Alloisoleucine
threo-3-Hydroxyleucine 5,5,5 -Trifluoroleucine Thialysine (S-2-aminoethylcysteine) 6-C-Methyllysine 5-Hydroxylysine
trans-4,5-Dehydrolysine
Methionine
Phenylalanine
Proline
2,6-Diamino-4-hexynoic acid 4-Oxalysine 4-Selenalysine Ethionine Selenomethionine cis -Crotylglycine Norleucine 2- Fluorophenylalanine 3-Fluorophenylalanine 4-Fluorophenylalanine fl-2-Thienylalanine /3-3-Thienylalanine 2,5-Dihydrophenylalanine 3-Phenylserine 3-Furyl-3-alanine m-Tyrosine 3,4-Dehydroproline Azetidine-2-carboxylic acid
cis-4~Fluoroproline trans -4-Fluoroproline 4-Thioproline 4-Selenaproline
cis-4-Hydroxyproline Threonine
3-Hydroxynorvaline
Concentration a (raM) 1 0.2 0.1 5 0.2 180 --1 -80 0.5 -0.3 15 0.5 -8 ----2 ---0.3 1 0.5 4 --4 -2 10 l0 --15 -20 1
References b 19 7, 20 20 20 21 22 23 24 25 26 27 28, 29 30 8 16 31 31 31 32 32 32 33 34 35 36 37 38 38 39 38 40 41 42 43 29 44 45 2 2 46 47 48 9, 49
(continued)
780
[61]
TARGETING: SELECTED TECHNIQUES AMINO ACID ANALOGS INCORPORATED INTO PROTEIN (continued)
Amino acid Tryptophan
Tyrosine Valine
Analog 7-Azatryptophan 4-Fluorotryptophan 5-Fluorotryptophan 6-Fluorotryptophan Tryptazan 4-Methyltryptophan m-Fluorotyrosine 3,4-Dihydroxyphenylalanine 2-Amino-3-chlorobutyric acid Cyclobutyglycine Cyclopropylglycine Penicillamine AUoisoleucine
Concentration" (mM) Referencesb Inactive --0.5 --0.5 5 0.1 --Inactive 70
50 51 51 52 53 54 12, 55 56 57 58 59 60 30
Concentration resulting in 50% inhibition of incorporation of the corresponding amino acid in the Krebs II ascites cell-free system described in Fig. 1. b Numbers refer to text footnotes. ple, trifluoroleucine was incorporated by bacteria, 16 but not by a eukaryotic system. 17 As a result, w h e n a particular analog is applied to a new system, it is n e c e s s a r y to assess critically whether the analog is incorporated into protein and to establish its effective concentration. Selection of Analog Concentration Selection of amino acids for incorporation into protein depends on the selectivity of a m i n o a c y l - t R N A synthetases. These e n z y m e s possess an extraordinary ability to discriminate a m o n g structurally similar amino acid substrates. ~5,18 Consequently, amino acid analogs generally are linked to t R N A m u c h less efficiently than their corresponding amino acids. As an example, Fig. I c o m p a r e s the competitive inhibition of [3H]threonine incorporation by threonine and a threonine analog. Assuming that only the L-isomer is active, the analog is 50 times less effective. As a result, to achieve efficient incorporation of the analog, high concentrations must be used, and the naturally occurring amino acid should be depleted as m u c h as possible. (Concentrations of analogs resulting in 50% inhibition are listed in the table. 2,7-9,12,16,19-6°) J60. M. Rennert and H. S. Anker, Biochemistry 2, 471 (1963). 17 O. M. Rennert and H. S. Anker, Nature (London) 203, 1256 (1964). ~8p. R. Schimmel and D. $611, Annu. Rev. Biochem. 48, 601 (1979). ~9 p. F. Kruse, P. B. White, H. A. Carter, and T. A. McCoy, Cancer Res. 19, 122 (1959).
[61]
AMINO ACID ANALOGS
781
i
15
xynorvahne
×
E
Q_ u
5
0
i i 10 100 1000 CONCENTRATION (~M)
10,000
FIG. 1. Inhibition of [3H]threonine incorporation by L-threonine or by DL-threo-fl-hydroxynorvaline. Varying amounts of these amino acids were added to the Krebs II ascites cell-free system containing 4/zM [3H]threonine (6 Ci/mmol). After incubation for 30 min, reactions were assayed for incorporation of label into tRNA and protein by spotting 20-~1 aliquots on Whatman 3 MM filter paper disks, rinsing the disks four times for l0 min in 5% trichloroacetic acid, and counting the dry filters. 64
z0 A. M. Stem, B. M. Foxman, A. H. Tashjian, Jr., and R. H. Abeles, J. Med. Chem. 25,544 (1982). 21 D. C. Klein, J. L. Weller, K. L. Kirk, and R. W. Hartley, Mol. Pharmacol. 13, 1105 (1977). 22 A. P. Levin and P. E. Hartman, J. Bacteriol. 86, 820 (1963). 23 S. Schlesinger and M. J. Schelsinger, J. Biol. Chem. 244, 3803 (1969). 24 A. K. Williams, S. T. Cox, and R. G. Eagon, Biochem. Biophys. Res. Commun. 18, 250 (1965). 25 T. J. McCord, D. C. Howell, D. L. Tharp, and A. L. Davis, J. Med. Chem. 8, 290 (1965). 26 C. B. Hiremath, G. Olson, and C. Rosenblum, Biochemistry 10, 1096 (1971). 27 M. E. Smulson and M. Rabinowitz, Arch. Biochem. Biophys. 124, 306 (1968). 28 H. Gershon, L. Shanks, and D. D. Clarke, J. Pharm. Sci. 67, 715 (1978). 29 G. Hortin and I. Boime, unpublished result. 3o R. B. Loftfield, L. I. Hecht, and E. A. Eigner, Biochim. Biophys. Acta 72, 383 (1963). 31 M. Rabinowitz and K. Tuve, Proc. Soc. Exp. Biol. Med. 100, 222 (1959). 32 E. M. Lansford, N. M. Lee, and W. Shive, Arch. Biochem. Biophys. 119, 272 (1967). 33 C. DeMarco, V. Busiello, M. DiGirolamo, and D. Cavallini, Biochim. Biophys. Acta 454, 298 (1976). 34 M. Levine and H. Tarver, J. Biol. Chem. 192, 835 (1951). 35 D. B. Cowie and G. N. Cohen, Biochim. Biophys. Acta 26, 252 (1957). 36 C. G. Skinner, J. Edelson, and W. Shive, J. Am. Chem. Soc. 83, 2281 (1961). 37 D. B. Cowie, G. N. Cohen, E. T. Bolton, and H. deRobichon-Szulmajster, Biochim. Biophys. Acta 34, 39 (1959). 38 W. L. Fangman and F. C. Niedhardt, J. Biol. Chem. 239, 1939 (1964). 39 R. Munier and G. N. Cohen, Biochim. Biophys. Acta 21, 592 (1956). 4o B. A. Samal, L. E. Frazier, G. Monto, A. Slesers, Z. Hruban, and R. W. Wissler, Proc. Soc. Exp. Biol. 112, 442 (1963).
782
TARGETING: SELECTED TECHNIQUES
[61]
Assay of tRNA charging as shown in Fig. 1 is a useful approach for estimating the minimal concentration of amino acid analog required in cell-free systems to achieve efficient incorporation of the analog. If the analog is incorporated into protein, it necessarily competes with a naturally utilized amino acid for aminoacylation of tRNA. However, it should be kept in mind that some compounds not linked to tRNA, such as amino alcohols 6j and adenylates of amino acids, 62 also inhibit aminoacylation of tRNA. Thus, this assay does not demonstrate incorporation of an analog into protein. As a control for nonspecific inhibition of aminoacylation, amino acid analogs should be assayed for effects on the charging of other amino acids. In the case of the threonine analog in Fig. 1, the highest concentration used did not block incorporation of methionine into aminoacyltRNA. Cell-free translation systems appear to tolerate concentrations of greater than 100 mM of many analogs, even though translational efficiency is very dependent on the concentration of some ions such as potassium and magnesium. 63 Neutral amino acids have an acidic isoelectric point so that solutions need to be adjusted with a base such as NaOH or 41 M. J. Pine, Antimicrob. Agents Chemother. 7, 601 (1975). 42 j. Janacek, J. Chaloupka, K. Veres, and M. Havranek, Nature (London) 184, 1895 (1959). 43 j. Janacek, Folia Mierobiol. (Prague) 12, 132 (1967). 44 j. Rosenbloom and D. J. Prockop, J. Biol. Chem. 245, 3361 (1970). 45 T. Takeuchi and D. J. Prockop, Biochim. Biophys. Acta 175, 142 (1969). 46 V. Busiello, Biochim. Biophys. Acta 564, 311 (1979). 47 V. Busiello, Biochim. Biophys. Acta 606, 347 (1980). 48 j. Rosenbloom and D. J. Prockop, J. Biol. Chem. 246, 1549 (1971). 49 p. Christner, A. Carpousis, M. Harsch, and J. Rosenbloom, J. Biol. Chem. 250, 7623 (1975). 5o A. B. Pardee, V. G. Shore, and L. S. Prestidge, Biochim. Biophys. Acta 21, 406 (1956). 51 D. T. Browne, G. L. Kenyon, and G. D. Hegemon, Biochem. Biophys. Res. Commun. 39, 13 (1970). 52 Ao B. Pardee and L. S. Prestidge, Biochim. Biophys. Acta 27, 330 (1958). 53 A. B. Pardee and L. S. Prestidge, Biochim. Biophys. Acta 21, 406 (1956). 54 R. D. Mosteller and C. Yanofsky, Fed. Proc., Fed. Am. Soc. Exp. Biol. 29, 598 (1970). 55 R. S. Schweet and E. H. Allen, J. Biol. Chem. 233, 1104 (1958). 56 R. Calendar and P. Berg, Biochemistry 5, 1690 (1966). 57 M. Freundlich, Science 157, 823 (1967). 58 T. H. Porter, S. C. Smith, and W. Shive, Arch. Biochem. Biophys. 179, 266 (1977). 59 W. M. Harding and M. L. DeShazo, Arch. Biochem. Biophys. 118, 23 (1967). 6o E. Lodemann, P. Ulrich, and A. Wacker, Biochim. Biophys. Acta 474, 210 (1977). 61 R. B. Loftfield, Prog. Nucleic Acid Res. Mol. Biol. 12, 87 (1972). 62 D. Cassio, F. Lemoine, J. Waller, E. Sandrin, and R. A. Boissonnas, Biochemistry 6, 827 (1967). 63 S. Daniels-McQueen, D. McWilliams, S. Birken, R. Canfield, T. Landefeld, and I. Boime, J. Biol. Chem. 253, 7109 (1978).
[61]
AMINO AClD ANALOGS
783
tris(hydroxymethyl)aminomethanebefore they are used. If an analog is an acidic or basic amino acid or it occurs as a hydrochloride salt, the maximum concentration may be limited by the counterions required for neutralization. Determination of the appropriate analog concentration for use with intact cells presents greater difficulty owing to the added complications of uptake into cells, amino acid metabolism, and endogenous amino acid pools. Competition assays as in Fig. 1 are of less use in these cases than in cell-free systems. In practice, several analog concentrations should be tried, and the maximal concentration that does not cause toxicity or excessive inhibition of protein synthesis should be used. Demonstration of Amino Acid Analog Incorporation into Protein Incorporation of an amino acid analog into protein can be shown most directly by use of radioactively labeled analogs. If incorporation of the label occurs, the labeled protein should be hydrolyzed and the hydrolyzate analyzed to ensure that the analog was not converted to different compounds before being incorporated. Other studies have detected the incorporation of unlabeled analogs by amino acid analysis of a protein hydrolyzate. More indirect evidence for the incorporation of amino acid analogs is provided by slight alteration of the electrophoretic mobility of proteins in polyacrylamide gels containing sodium dodecyl sulfate. 8 This can be examined very easily, but it is not complete proof of incorporation. Mobility could be changed by altered protein processing. Also, if an analog induces starvation for a particular amino acid, other amino acids may be substituted for it in protein--a phenomenon that has been termed stuttering. 64 Analysis of the Effects of Amino Acid Analogs on Protein Processing If an amino acid analog affects the processing of a protein, the most basic issue is whether the effect results from incorporation of the analog into the protein of interest. Several criteria, besides demonstration that the analog is capable of incorporation into protein, are helpful. 1. The effective analog concentration equals the concentration required for analog incorporation. 2. The effect should be blocked specifically by the amino acid replaced by the analog. 64 j. Parker, J. W. Pollard, J. D. Friesen, and C. P. Stanners, Proc. Natl. Acad. Sci. U.S.A. 75, 1091 (1978).
784
TARGETING" SELECTED TECHNIQUES
[62]
3. The effect should appear with brief exposure to the analog. If cells are exposed to analogs for many hours, the protein processing mechanism may be altered, and the levels of many enzymes may change because incorporation of analogs generally increases the turnover of proteins and increases their susceptibility to protea s e s 65 Acknowledgments We thank Drs. Robert Abeles, Herman Gershon, Robert Handschumacher, Kenneth Klein, Theodore Otani, Upendra Pandit, and Marco Rabinowitz for supplying amino acid analogs for our studies. 65 A. Goldberg and A. C. St. John, Annu. Rev. Biochem. 45, 747 (1976).
[62] Q u a n t i t a t i v e A s s a y for Signal P e p t i d a s e 1 By ROBERT C. JACKSON
Signal peptidase is the enzyme responsible for removing the signal peptide portion of nascent presecretory and presumably also prelysosomal and premembrane proteins during their cotranslational translocation across the rough-endoplasmic reticulum (RER) membrane. In many respects signal peptidase is a unique protease. It is an integral membrane protein whose substrate is not a full-length protein, but, rather, an incomplete polypeptide chain that is engaged in the translocation process. These unique properties of signal peptidase were the source of several obstacles to its assay. Signal peptidase activity was first detected by a cotranslational, or translocation-dependent assay. 2 In this assay the entire series of events that occurs at the RER membrane during translocation is reconstituted in vitro. The assay involves the translation of mRNA encoding a secretory protein in a cell-free in vitro translation system containing microsomal membranes. Polysomes bearing nascent presecretory proteins bind to elements on the cytoplasmic surface of the microsomes, and the nascent presecretory proteins are translocated across the microsomal membrane. During translocation across the membrane they become available to sigThis work was supported by United States Public Health Service Grant GM26763. 2 G. Blobel and B. Dobberstein, J. Cell Biol. 67, 852 (1975).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
AMINO
ACID ANALOGS
777
6). Furthermore, it was determined that the sizes of the cotranslationally modified lysosomal polypeptides were unaffected by the proteases unless detergent was added, in which case digestion proceeded to yield acidsoluble fragments, indicating that the polypeptide had been completely transferred across the membrane and segregated into the microsomal lumen, as is the case with secretory proteins. 22 22 G. Blobel and B. Dobberstein, J. Cell Biol. 67, 835 (1975).
[61] A p p l i c a t i o n s o f A m i n o A c i d A n a l o g s for S t u d y i n g Coand Posttranslational Modifications of Proteins B y G L E N H O R T I N a n d IRVING B O I M E
Synthesis of proteins often requires modification of the primary translation products, and numerous types of covalent modification of peptide chains have been observed. These changes can occur on the protein during its synthesis (cotranslational) or after release from the ribosome (posttranslational). Understanding the assembly of mature proteins requires definition of the mechanisms of these reactions and of the structural determinants that limit them to particular sites in the cellular environment. One approach to this question is to incorporate amino acid analogs into protein and to examine the consequent effects on processing. There are several examples of such applications of amino acid analogs. Baltimore and Jacobson ~used them to inhibit the proteolytic cleavage of viral polyproteins and thus establish that all the peptides were derived from a single precursor. Hydroxyproline synthesis in collagen was probed with proline analogs,2 clarifying the mechanism of this hydroxylation reaction. Proteolytic cleavage of prosomatostatin, proinsulin, 3 and pro-opiomelanocortin4,5 were inhibited by analogs of arginine and lysine. Asparagine-linked glycosylation was inhibited by analogs of threonine 6 and asparagine, 7 yielding insights into the specificity and mechanism of this reaction. Analogs of i M. F. Jacobson and D. Baltimore, Proc. Natl. Acad. Sci. U.S.A. 61, 77 (1968). 2 A. A. Gottlieb, Y. Fujita, S. Udenfriend, and B. Witkop, Biochemistry 4, 2507 (1965). 3 B. D. Noe, J. Biol. Chem. 256, 4940 (1981). 4 p. Crine and E. Lemieux, J. Biol. Chem. 257, 832 (1982). 5 H. Hoshina, G. Hortin, and I. Boime, Science 217, 63 (1982). 6 G. Hortin and I. Boime, J. Biol. Chem. 255, 8007 (1980). 7 G. Hortin, I. Boime, A. M. Stern, B. Miller, and R. H. Abeles, J. Biol. Chem. 257, 4047 (1983).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5
778
TARGETING: SELECTED TECHNIQUES
[61]
leucine and threonine have been useful for probing the process by which secretory proteins traverse the membrane of the endoplasmic reticulum 8-~° and for examining the specificity of the protease that excises amino-terminal prepeptides from most secretory proteins.)1 The diversity of these examples illustrates the versatility of this approach. Virtually any process dependent on protein structure can be examined in this manner. Use of amino acid analogs has several characteristics that are often advantageous. 1. Proteins are modified cotranslationally, permitting analysis of modification reactions that occur on nascent peptide chains. 2. Subtle modifications of protein structure can be introduced because most analogs are structurally very similar to the amino acids they supplant. 3. Protein can be labeled with new heteroatoms such as selenium, chlorine, and fluorine. Fluorine, in particular, has been useful as a probe for nuclear magnetic resonance (NMR) spectroscopy of proteins. 12 4. They can be used in any system--intact organisms, organ culture, cell culture, and cell-free systems. Owing to the great variety of applications of this approach several general considerations are presented here rather than a detailed protocol with restricted applicability. Selection of Amino Acid Analogs A large number of amino acid analogs have been reported to be incorporated into protein. Some of these compounds are listed in the table. Analogs probably can be found to replace all the amino acids with the possible exception of glycine, alanine, and serine. A large number of potential analogs have not been studied thoroughly. Numerous additional amino acid antagonists are listed in other references. J3-j5 Some analogs listed in the table may be effective only in particular systems. For exams G.
Hortin and I. Boime, Proc. Natl. Acad. Sci. U.S.A. 77, 1356 (1980). 9 G. Hortin and I. Boime, J. Biol. Chem. 255, 7051 (1980). ~0p. Walter, I. Ibrahimi, and G. Blobel, J. Cell Biol. 91, 545 (1981). 11 G. Hortin and I. Boime, Cell 24, 453 (1981). 12 B. D. Sykes, H. I. Weingarten, and M. J. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 71, 469 (1974). 13 K. Dittmer, Ann. N . Y . Acad. Sci. 52, 1274 (1950). 14 W. Shive and C. G. Skinner, in "Metabolic Inhibitors" (R. M. Hochster and J. H. Quastel, eds.), p. 2. Academic Press, New York, 1963. ~5G. L. Igloi, F. yon der Haar, and F. Cramer, Biochemistry 17, 3459 (1978).
[61]
AMINO ACID ANALOGS
779
AMINO ACID ANALOGS INCORPORATED INTO PROTEIN
Amino acid Arginine Asparagine Aspartic acid Histidine
Isoleucine
Leucine Lysine
Analog Canavanine
threo-3-Fluoroasparagine threo-3-Fluoroaspartic acid erythro-3-Fluoroaspartic acid 2-Fluorohistidine 1,2,4-Triazole-3-alanine 2-Methylhistidine 3-Amino- 1,2,4-triazoylalanine 4-Thiaisoleucine O-Ethylthreonine O-Methylthreonine 4-Fluoroisoleucine Alloisoleucine
threo-3-Hydroxyleucine 5,5,5 -Trifluoroleucine Thialysine (S-2-aminoethylcysteine) 6-C-Methyllysine 5-Hydroxylysine
trans-4,5-Dehydrolysine
Methionine
Phenylalanine
Proline
2,6-Diamino-4-hexynoic acid 4-Oxalysine 4-Selenalysine Ethionine Selenomethionine cis -Crotylglycine Norleucine 2- Fluorophenylalanine 3-Fluorophenylalanine 4-Fluorophenylalanine fl-2-Thienylalanine /3-3-Thienylalanine 2,5-Dihydrophenylalanine 3-Phenylserine 3-Furyl-3-alanine m-Tyrosine 3,4-Dehydroproline Azetidine-2-carboxylic acid
cis-4~Fluoroproline trans -4-Fluoroproline 4-Thioproline 4-Selenaproline
cis-4-Hydroxyproline Threonine
3-Hydroxynorvaline
Concentration a (raM) 1 0.2 0.1 5 0.2 180 --1 -80 0.5 -0.3 15 0.5 -8 ----2 ---0.3 1 0.5 4 --4 -2 10 l0 --15 -20 1
References b 19 7, 20 20 20 21 22 23 24 25 26 27 28, 29 30 8 16 31 31 31 32 32 32 33 34 35 36 37 38 38 39 38 40 41 42 43 29 44 45 2 2 46 47 48 9, 49
(continued)
780
[61]
TARGETING: SELECTED TECHNIQUES AMINO ACID ANALOGS INCORPORATED INTO PROTEIN (continued)
Amino acid Tryptophan
Tyrosine Valine
Analog 7-Azatryptophan 4-Fluorotryptophan 5-Fluorotryptophan 6-Fluorotryptophan Tryptazan 4-Methyltryptophan m-Fluorotyrosine 3,4-Dihydroxyphenylalanine 2-Amino-3-chlorobutyric acid Cyclobutyglycine Cyclopropylglycine Penicillamine AUoisoleucine
Concentration" (mM) Referencesb Inactive --0.5 --0.5 5 0.1 --Inactive 70
50 51 51 52 53 54 12, 55 56 57 58 59 60 30
Concentration resulting in 50% inhibition of incorporation of the corresponding amino acid in the Krebs II ascites cell-free system described in Fig. 1. b Numbers refer to text footnotes. ple, trifluoroleucine was incorporated by bacteria, 16 but not by a eukaryotic system. 17 As a result, w h e n a particular analog is applied to a new system, it is n e c e s s a r y to assess critically whether the analog is incorporated into protein and to establish its effective concentration. Selection of Analog Concentration Selection of amino acids for incorporation into protein depends on the selectivity of a m i n o a c y l - t R N A synthetases. These e n z y m e s possess an extraordinary ability to discriminate a m o n g structurally similar amino acid substrates. ~5,18 Consequently, amino acid analogs generally are linked to t R N A m u c h less efficiently than their corresponding amino acids. As an example, Fig. I c o m p a r e s the competitive inhibition of [3H]threonine incorporation by threonine and a threonine analog. Assuming that only the L-isomer is active, the analog is 50 times less effective. As a result, to achieve efficient incorporation of the analog, high concentrations must be used, and the naturally occurring amino acid should be depleted as m u c h as possible. (Concentrations of analogs resulting in 50% inhibition are listed in the table. 2,7-9,12,16,19-6°) J60. M. Rennert and H. S. Anker, Biochemistry 2, 471 (1963). 17 O. M. Rennert and H. S. Anker, Nature (London) 203, 1256 (1964). ~8p. R. Schimmel and D. $611, Annu. Rev. Biochem. 48, 601 (1979). ~9 p. F. Kruse, P. B. White, H. A. Carter, and T. A. McCoy, Cancer Res. 19, 122 (1959).
[61]
AMINO ACID ANALOGS
781
i
15
xynorvahne
×
E
Q_ u
5
0
i i 10 100 1000 CONCENTRATION (~M)
10,000
FIG. 1. Inhibition of [3H]threonine incorporation by L-threonine or by DL-threo-fl-hydroxynorvaline. Varying amounts of these amino acids were added to the Krebs II ascites cell-free system containing 4/zM [3H]threonine (6 Ci/mmol). After incubation for 30 min, reactions were assayed for incorporation of label into tRNA and protein by spotting 20-~1 aliquots on Whatman 3 MM filter paper disks, rinsing the disks four times for l0 min in 5% trichloroacetic acid, and counting the dry filters. 64
z0 A. M. Stem, B. M. Foxman, A. H. Tashjian, Jr., and R. H. Abeles, J. Med. Chem. 25,544 (1982). 21 D. C. Klein, J. L. Weller, K. L. Kirk, and R. W. Hartley, Mol. Pharmacol. 13, 1105 (1977). 22 A. P. Levin and P. E. Hartman, J. Bacteriol. 86, 820 (1963). 23 S. Schlesinger and M. J. Schelsinger, J. Biol. Chem. 244, 3803 (1969). 24 A. K. Williams, S. T. Cox, and R. G. Eagon, Biochem. Biophys. Res. Commun. 18, 250 (1965). 25 T. J. McCord, D. C. Howell, D. L. Tharp, and A. L. Davis, J. Med. Chem. 8, 290 (1965). 26 C. B. Hiremath, G. Olson, and C. Rosenblum, Biochemistry 10, 1096 (1971). 27 M. E. Smulson and M. Rabinowitz, Arch. Biochem. Biophys. 124, 306 (1968). 28 H. Gershon, L. Shanks, and D. D. Clarke, J. Pharm. Sci. 67, 715 (1978). 29 G. Hortin and I. Boime, unpublished result. 3o R. B. Loftfield, L. I. Hecht, and E. A. Eigner, Biochim. Biophys. Acta 72, 383 (1963). 31 M. Rabinowitz and K. Tuve, Proc. Soc. Exp. Biol. Med. 100, 222 (1959). 32 E. M. Lansford, N. M. Lee, and W. Shive, Arch. Biochem. Biophys. 119, 272 (1967). 33 C. DeMarco, V. Busiello, M. DiGirolamo, and D. Cavallini, Biochim. Biophys. Acta 454, 298 (1976). 34 M. Levine and H. Tarver, J. Biol. Chem. 192, 835 (1951). 35 D. B. Cowie and G. N. Cohen, Biochim. Biophys. Acta 26, 252 (1957). 36 C. G. Skinner, J. Edelson, and W. Shive, J. Am. Chem. Soc. 83, 2281 (1961). 37 D. B. Cowie, G. N. Cohen, E. T. Bolton, and H. deRobichon-Szulmajster, Biochim. Biophys. Acta 34, 39 (1959). 38 W. L. Fangman and F. C. Niedhardt, J. Biol. Chem. 239, 1939 (1964). 39 R. Munier and G. N. Cohen, Biochim. Biophys. Acta 21, 592 (1956). 4o B. A. Samal, L. E. Frazier, G. Monto, A. Slesers, Z. Hruban, and R. W. Wissler, Proc. Soc. Exp. Biol. 112, 442 (1963).
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Assay of tRNA charging as shown in Fig. 1 is a useful approach for estimating the minimal concentration of amino acid analog required in cell-free systems to achieve efficient incorporation of the analog. If the analog is incorporated into protein, it necessarily competes with a naturally utilized amino acid for aminoacylation of tRNA. However, it should be kept in mind that some compounds not linked to tRNA, such as amino alcohols 6j and adenylates of amino acids, 62 also inhibit aminoacylation of tRNA. Thus, this assay does not demonstrate incorporation of an analog into protein. As a control for nonspecific inhibition of aminoacylation, amino acid analogs should be assayed for effects on the charging of other amino acids. In the case of the threonine analog in Fig. 1, the highest concentration used did not block incorporation of methionine into aminoacyltRNA. Cell-free translation systems appear to tolerate concentrations of greater than 100 mM of many analogs, even though translational efficiency is very dependent on the concentration of some ions such as potassium and magnesium. 63 Neutral amino acids have an acidic isoelectric point so that solutions need to be adjusted with a base such as NaOH or 41 M. J. Pine, Antimicrob. Agents Chemother. 7, 601 (1975). 42 j. Janacek, J. Chaloupka, K. Veres, and M. Havranek, Nature (London) 184, 1895 (1959). 43 j. Janacek, Folia Mierobiol. (Prague) 12, 132 (1967). 44 j. Rosenbloom and D. J. Prockop, J. Biol. Chem. 245, 3361 (1970). 45 T. Takeuchi and D. J. Prockop, Biochim. Biophys. Acta 175, 142 (1969). 46 V. Busiello, Biochim. Biophys. Acta 564, 311 (1979). 47 V. Busiello, Biochim. Biophys. Acta 606, 347 (1980). 48 j. Rosenbloom and D. J. Prockop, J. Biol. Chem. 246, 1549 (1971). 49 p. Christner, A. Carpousis, M. Harsch, and J. Rosenbloom, J. Biol. Chem. 250, 7623 (1975). 5o A. B. Pardee, V. G. Shore, and L. S. Prestidge, Biochim. Biophys. Acta 21, 406 (1956). 51 D. T. Browne, G. L. Kenyon, and G. D. Hegemon, Biochem. Biophys. Res. Commun. 39, 13 (1970). 52 Ao B. Pardee and L. S. Prestidge, Biochim. Biophys. Acta 27, 330 (1958). 53 A. B. Pardee and L. S. Prestidge, Biochim. Biophys. Acta 21, 406 (1956). 54 R. D. Mosteller and C. Yanofsky, Fed. Proc., Fed. Am. Soc. Exp. Biol. 29, 598 (1970). 55 R. S. Schweet and E. H. Allen, J. Biol. Chem. 233, 1104 (1958). 56 R. Calendar and P. Berg, Biochemistry 5, 1690 (1966). 57 M. Freundlich, Science 157, 823 (1967). 58 T. H. Porter, S. C. Smith, and W. Shive, Arch. Biochem. Biophys. 179, 266 (1977). 59 W. M. Harding and M. L. DeShazo, Arch. Biochem. Biophys. 118, 23 (1967). 6o E. Lodemann, P. Ulrich, and A. Wacker, Biochim. Biophys. Acta 474, 210 (1977). 61 R. B. Loftfield, Prog. Nucleic Acid Res. Mol. Biol. 12, 87 (1972). 62 D. Cassio, F. Lemoine, J. Waller, E. Sandrin, and R. A. Boissonnas, Biochemistry 6, 827 (1967). 63 S. Daniels-McQueen, D. McWilliams, S. Birken, R. Canfield, T. Landefeld, and I. Boime, J. Biol. Chem. 253, 7109 (1978).
[61]
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783
tris(hydroxymethyl)aminomethanebefore they are used. If an analog is an acidic or basic amino acid or it occurs as a hydrochloride salt, the maximum concentration may be limited by the counterions required for neutralization. Determination of the appropriate analog concentration for use with intact cells presents greater difficulty owing to the added complications of uptake into cells, amino acid metabolism, and endogenous amino acid pools. Competition assays as in Fig. 1 are of less use in these cases than in cell-free systems. In practice, several analog concentrations should be tried, and the maximal concentration that does not cause toxicity or excessive inhibition of protein synthesis should be used. Demonstration of Amino Acid Analog Incorporation into Protein Incorporation of an amino acid analog into protein can be shown most directly by use of radioactively labeled analogs. If incorporation of the label occurs, the labeled protein should be hydrolyzed and the hydrolyzate analyzed to ensure that the analog was not converted to different compounds before being incorporated. Other studies have detected the incorporation of unlabeled analogs by amino acid analysis of a protein hydrolyzate. More indirect evidence for the incorporation of amino acid analogs is provided by slight alteration of the electrophoretic mobility of proteins in polyacrylamide gels containing sodium dodecyl sulfate. 8 This can be examined very easily, but it is not complete proof of incorporation. Mobility could be changed by altered protein processing. Also, if an analog induces starvation for a particular amino acid, other amino acids may be substituted for it in protein--a phenomenon that has been termed stuttering. 64 Analysis of the Effects of Amino Acid Analogs on Protein Processing If an amino acid analog affects the processing of a protein, the most basic issue is whether the effect results from incorporation of the analog into the protein of interest. Several criteria, besides demonstration that the analog is capable of incorporation into protein, are helpful. 1. The effective analog concentration equals the concentration required for analog incorporation. 2. The effect should be blocked specifically by the amino acid replaced by the analog. 64 j. Parker, J. W. Pollard, J. D. Friesen, and C. P. Stanners, Proc. Natl. Acad. Sci. U.S.A. 75, 1091 (1978).
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[62]
3. The effect should appear with brief exposure to the analog. If cells are exposed to analogs for many hours, the protein processing mechanism may be altered, and the levels of many enzymes may change because incorporation of analogs generally increases the turnover of proteins and increases their susceptibility to protea s e s 65 Acknowledgments We thank Drs. Robert Abeles, Herman Gershon, Robert Handschumacher, Kenneth Klein, Theodore Otani, Upendra Pandit, and Marco Rabinowitz for supplying amino acid analogs for our studies. 65 A. Goldberg and A. C. St. John, Annu. Rev. Biochem. 45, 747 (1976).
[62] Q u a n t i t a t i v e A s s a y for Signal P e p t i d a s e 1 By ROBERT C. JACKSON
Signal peptidase is the enzyme responsible for removing the signal peptide portion of nascent presecretory and presumably also prelysosomal and premembrane proteins during their cotranslational translocation across the rough-endoplasmic reticulum (RER) membrane. In many respects signal peptidase is a unique protease. It is an integral membrane protein whose substrate is not a full-length protein, but, rather, an incomplete polypeptide chain that is engaged in the translocation process. These unique properties of signal peptidase were the source of several obstacles to its assay. Signal peptidase activity was first detected by a cotranslational, or translocation-dependent assay. 2 In this assay the entire series of events that occurs at the RER membrane during translocation is reconstituted in vitro. The assay involves the translation of mRNA encoding a secretory protein in a cell-free in vitro translation system containing microsomal membranes. Polysomes bearing nascent presecretory proteins bind to elements on the cytoplasmic surface of the microsomes, and the nascent presecretory proteins are translocated across the microsomal membrane. During translocation across the membrane they become available to sigThis work was supported by United States Public Health Service Grant GM26763. 2 G. Blobel and B. Dobberstein, J. Cell Biol. 67, 852 (1975).
METHODS IN ENZYMOLOGY, VOL. 96
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181996-5