ENCYCLOPEDIA OF
`
`MOLECULAR
`BIOLOGY
`
`VOLUME 1
`
`Thomas E. Creighton
`European Molecular Biology Laboratory
`London, England
`
`A Wiley-lnterscience Publication
`John Wiley & Sons, Inc.
`New York/ Chichester/ Weinheim /Brisbane/ Singapore/ Toronto
`
`1 of 46
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`This book is printed on acid-free paper. @
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`Copyright © I 999 by John Wiley & Sons, Inc.
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`All rights reserved. Published simultaneously in Canada.
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`No part of this publication may be reproduced, stored in a retrieval system or 1ra11smitted
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`
`library of Congress Cataloging-in-Publication Data:
`
`Creighton, Thomas E., 1940-
`The encyclopedia of molecular biology / Thomas E. Creighton.
`p. cm.
`Includes index.
`ISBN 0-471- 15302-8 (alk. paper)
`I. Molecular biology- Encyclopedias.
`QH506.C74 1999
`572.8' 03-dc2 I
`
`I. Title.
`
`Printed in the United States of America.
`
`10 9 8 7 6 5 4 3 2
`
`Un1v.-::rs -
`
`99-11575
`cw
`
`·']\ Library'-.
`..'1-i'.'ladisqrt'
`
`2 of 46
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`72
`
`AFFINITY CHROMATOGRAPHY
`
`25. G. Ughetto (1988) In Anthracycline and Anthracenedione-Based
`Anticancer Agents (J. W. Lown, ed.), Elsevier, Amsterdam,
`pp. 295-334.
`26. F. Barcelo, J. Martorell, F. Gavilanes, and J. M. Gonzalez-Ros
`(1988)Biochem. Pharmacol. 37, 2133-2138.
`27. E. Stutter, H. Schuetz, and H. Berg (1988) In Anthracycline and
`Anthracenedione-Based Anticancer Agents (J. W. Lown, ed.), El(cid:173)
`sevier, Amsterdam, pp. 245-'293.
`28. K Chen, N. Gresh, and B. Pullman (1986) Nucleic Acids Res. 14,
`2251-2267.
`29. B. Pullman (1991)Anti-Cancer Drug Design 7, 95-105.
`30. H. Trist and D. R. Phillips (1989) Nucleic Acids Res. 17, 3673-
`3688.
`31. B. Gandecha and J. R. Brown (1985) Biochem. Pharmacol. 34,
`733-736.
`32. D. R. Phillips, P. Greif, and R. C. Boston (1988) Molec. Pharmacol.
`33, 225-230.
`33. Ref. 14, pp. 528-569.
`34. C. E. Myers (1992) Cancer Chemother. Biol. Resp. Modifiers Ann.
`13, 45-52.
`35. Ref. 14, pp. 546-551.
`36. M. Gigli, S. M. Doglia, J . M. Millot, L. Valantini, and M. Manfait
`(1988) Biochim. Biophys. Acta 950, 13- 20.
`37. J . Cummings and C. S. McArdle (1986) Br. J . Cancer 53, 835-
`838.
`38. L. Valentini, V. Nicolella, E. Vannini, M. Menozzi, S. Penco, and
`F. Arcamone (1985) IL. Farmaco Ed. Sci. 40, 377-389.
`39. C. Holm, J. Covey, D. Kerrigan, K W. Kohn, and Y. Pommier
`(1991) In DNA Topoisomerases in Cancer (M. Pomesil and
`K Kohn, eds.), Oxford University Press, New York, pp. 161-171.
`40. Y : Pommier (1995) In Anthracycline Antibiotics: New Analogues,
`Methods of Delivery and Mechanisms of Action, ACS Symposium
`Series No 574, pp. 183-203.
`41. G. J . Goldenberg, H. Wang, and G. W. Blair (1986) Cancer Res. 46,
`2978-2983.
`42. F. A. Fornari, W. D. Jarvis. M. S. Orr, J . K Randolph, S. Grant,
`and D. A. Gerwitz (1996) Biochem. Pharmacol. 51, 931-940.
`43. M. Binaschi, G. Capranico, P. De Isabella, M. Marini, R. Supino,
`and S. Tinelli (1990) Int. J. Cancer 45, 347-352.
`44. M. Binaschi, G. Capranico, L. Dal Bo, and F. Zunino (1997) Mol.
`Pharmacol; 51, 1053-1059.
`45. D. R. Phillips (1990) In Molecular Basis of Specificity in Nucleic
`Acid- Drug Interactions (B. Pullman and J . Jortner, eds.), Kluwer
`Academic, Dordrecht, The Netherlands, pp. 137-155.
`46. J. Cummings, L. Anderson, N. Willmott, and J. F. Smyth (1991)
`Eur. J. Cancer 27, 532-535.
`47. C. Cullinane and D. R. Phillips (1990) Biochemistry 29, 5638-
`5646.
`48. C. Cullinane, A. van Rosm~len, and D. R. Phillips (1994) Biochem(cid:173)
`istry 33, 4632-4638.
`49. S. M. Cutts and D. R. Phillips (1995) Nucleic Acids Res. 23, 2450-
`2456.
`50. A. van Rosmalen, C. Cullinane, S. M. Cutts, and D. R. Phillips
`(1995) Nucleic Acids Res. 23, 42-50.
`51. D. J . Taatjes, G. Guadiano, K Resing, and T. H. Koch (1996)
`J . Med. Chem. 39, 4135-4138.
`52. D. J. Taatjes, G. Guadiano, K Resing, and T. H. Koch (1997)
`J. Med. Chem. 40, 1276-1286.
`53. S. M. Zeman, D. R. Phillips, and C. M. Crothers (1998) Proc. Natl.
`Acad. Sci. USA 35, 11561-11565.
`54. A. Skladanowski and J . Konopa (1994) Biochem. Pharmacol. 47,
`2269-2278.
`
`55. A. Skladanowski and J . Konopa (1994) Biochem. Pharmacol. 47,
`2279-2287.
`56. C. Cullinane, S. M. Cutts, C. Panousis, and D. R. Phillips (1998)
`Proc. Am. Assoc. Cancer Res. 39, 424.
`57. G. Ciarrochi, M. Lestingi, M. Fontana, S. Spadari, and A. Monte(cid:173)
`cucco (1992) Biochem J. 279, 141-146.
`58. N. R. Bachur, R. Johnson, F. Yu, R. Hickey, N. Appelgren, and
`L. Malkas (1993) Mol. Pharmacol. 44, 1064-1069.
`59. G. Zaleskis, E . Berleth, S. Verstovek, M. J . Ehrke, and E. Mihich
`(1994) Mol. Pharmacol. 46, 901-908.
`60. A. Skladanowski and J . Konopa (1993) Biochem. Pharmacol. 46,
`375-382.
`61. R. B. Lock and L. Stribinskiene (1996) Cancer Res. 56, 4006.
`
`Suggestion• for Further Reading
`C. E. Myers, E. G. Mimnaugh, G. C. Yeh, and B. K Sinha (1988)
`Biochemical Mechanisms of Tumor Cell Kill by the Anthracyclines.
`In Anthracycline and Anthracenedione-Based Anticancer Agents
`(J. W. Lown, ed.), Elsevier, Amsterdam, pp. 527-569. (Contains
`an excellent discussion of the criteria for proof of the mechanism
`of action of Adriamycin and why it is difficult to prove how any
`anticancer drug kills cells.)
`R. B. Weiss (1992) The anthracyclines: Will we ever find a better dox(cid:173)
`orubicin? Semin. Oncol. 19, 670-686. (A most comprehensive re(cid:173)
`view of the history of Adriamycin, of the search for new derivatives,
`and of the clinical status of those derivatives.)
`W. B. Pratt, R. W. Ruddon, W. D. Ensminger, and J . Maybaum (1994)
`The Anticancer Drugs, 2nd ed., Oxford University Press, New York.
`B. A. Chabner and C. E. Myers (1993) In Cancer: Principles and Prac(cid:173)
`tice of Oncology (V. T. De Vita, S. Hellman, and S. A. Rosenberg,
`eds.), Lippincott, pp. 376-381. (An excellent concise review of Adri(cid:173)
`amycin, with an emphasis on cellular and clinical aspects.)
`
`AFFINITY CHROMATOGRAPHY
`
`SHMUEL SHALTIEL
`
`THE BASIC PRINCIPLE
`
`Affinity chromatography (AC) is a general chromatographic
`method for the selective extraction and purification of biologi(cid:173)
`cal macromolecules on the basis of their biorecognition (1-3).
`The method makes use of the specific physiological affinity be(cid:173)
`tween a desired macromolecule (M) and one of its physiologi(cid:173)
`cal ligands (L). The ligand, or its analogue (L'), actually acts
`as a "bait" and is used to extract or "fish out" a desired macro(cid:173)
`molecule (M1) (Fig. 1) from a mixture of macromolecules (M1;
`M 2; Ma; M4; M 5; Me ... ). The other macromolecules have a very
`low (if any) affinity for L, presumably because they are de(cid:173)
`signed to refrain from interfering in vivo with the physiolo~i(cid:173)
`cal recognition ofL by M.
`
`GENERAL PROCEDURE FOR AN AC PURIFICATION: KEY STEPS
`
`I. Immobilization (anchoring) of the ligand on an inert carrier:
`L is anchored on a carrier to yield an insoluble material, usu(cid:173)
`ally in a beaded form. This carrier should be as inert as possi(cid:173)
`ble (eg, beaded agarose) to achieve true active-site-mediated
`AC. Also, the attachment point of the ligand should not involve
`
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`/
`
`Selection
`adsorption
`
`lWashing
`
`Elution
`
`/
`
`I
`I
`I
`
`I 0!
`('\0'
`+~©
`Displac~ @
`
`1
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`
`Native
`(ligand free)
`M1
`
`Deformed
`M1
`
`Native
`(refolded)
`M1
`
`Figure 1. Schematic representation of the general procedure
`for an AC purification. For further details, see text.
`
`groups that are involved in binding to the macromolecule. Over
`the years, different ca~iers and various methods for ligand im(cid:173)
`mobilization were developed. These were reviewed and evalu(cid:173)
`ated by Wilchek et al. (3). In general, the anchoring of L onto
`an inert carrier involves (i) the introduction of chemically re(cid:173)
`active groups to the inert carrier, (ii) the covalent attachment
`ofL to the activated carrier, and (iii) inactivation of the excess
`of reactive groups (if any) that may remain on the activated
`carrier after completion of the ligand anchoring step. The im(cid:173)
`mobilized ligand can be used either batchwise or as a column.
`It may also find other uses-for example, to detect or demon(cid:173)
`strate specific protein-protein interactions by the binding
`of a specific protein. Furthermore, it may use the resulting col(cid:173)
`umn material to bind and fish out another protein that inter(cid:173)
`acts with it. Such immobilized ligands have been used also for
`labeling of cells, for the localization of proteins on cell surfaces,
`for the demonstration ofleakage of enzymes or specific proteins
`from damaged tissues, and so on.
`Historically, the pioneering work of Axen et al. (4) on the
`CNBr activation of beaded agarose had a great influence on
`the development of AC and the conversion of this methodol(cid:173)
`ogy into a most widely used tool in separation science. To this
`day, beaded agarose continues to be the inert carrier of choice,
`
`AFFINITY CHROMATOGRAPHY
`
`73
`
`and its activation with CNBr for ligand binding is still an ac(cid:173)
`tivation method of choice. A thorough analytical study of the
`mechanism of activation of agarose by CNBr (5) showed that
`three major products are formed: a carbamate (chemically in(cid:173)
`ert), a linear or a cyclic imidocarbonate (slightly reactive), and
`a cyanate ester (chemically very reactive). Analysis of freshly
`activated agarose showed that 60% to 85% of the total coupling
`capacity of the agarose is due to the formation of the cyanate
`esters. They are the ones that actually react and immobilize
`the ligand (Fig. 2). On the basis of this mechanism of activa(cid:173)
`tion by CNBr, it became possible to develop more efficient ac(cid:173)
`tivation procedures, which are reviewed in Refs. 3 and 6.
`2. Selective adsorption. The key selective step in AC is ob(cid:173)
`viously the extraction of the desired macromolecule M, which
`is singled out and removed by the immobilized L, from the
`mixture in which it is present. The macromolecule-be it an
`enzyme, an antibody, a receptor, a hormone, a growth fac(cid:173)
`tor, or the like-is selectively bound by the biospecific ligand
`L, which can be another protein, a peptide, a polynucleotide or
`a nucleotide, a polysacharide or a carbohydrate, a lipid, a vita(cid:173)
`min, or just a metal ion. Functionally, L may be a substrate, a
`substrate analogue, an inhibitor, an antigen, a coenzyme, a co(cid:173)
`factor, or a regulatory metabolite. In many cases, the biospe(cid:173)
`cific ligand used for the immobilization is a structural ana(cid:173)
`logue of the physiological ligand {L'). It is imperative, however,
`to ensure that it still retains the property of selective binding to
`M, and ideally to M only. In choosing the ligand for an affinity
`chromatography column, it is often possible to aqjust the grip
`ofM onto the anchored L, and thus to optimize both the adsorp(cid:173)
`tion and the elution steps. It should be noted that the adsorp(cid:173)
`tion conditions used (buffer, pH, ionic strength, temperatu.re)
`should also be carefully chosen to secure an optimal and selec(cid:173)
`tive adsorption.
`3. Washing out nonspecifically bound impurities. 'f!:us
`is usually carried out with an excess of the buffer used for
`selective adsorption.
`4. Elution of the desired macromolecule. The detachment of
`Mfrom the column (elution) is one of the most important steps
`in purification by AC. Obviously, the ideal elution is by a spe(cid:173)
`cific displacement of M with an excess of its biospecific ligand
`(Fig. 1). This procedure preserves the native structure of M
`by forming the more stable complex of M with its biospecific
`ligand L. When such elution is achieved, it strongly suggests
`that true active-site-mediated AC is involved. However, very
`often biospecific ligands fail to elute the desired protein, and
`nonspecific means have to be applied. These usually include
`a change in solvent or buffer composition, a change in pH or
`in ionic strength, the addition of a chaotropic or a "deform(cid:173)
`ing" buffer, a change in temperature, or a change in the electric
`field (electrophoretic desorption) (3). All these bring about a
`deformation of the protein (7,8), a concomitant loosening of the
`grip of M for L, and consequently elution. In some cases, the
`binding of M to the L column is so tight that it is not possible
`to recover Min a fully active form. IfM is an enzyme, this may
`yield a less active preparation (part of the M molecules may
`be totally inactive, or all molecules may have a lower affinity
`for the substrate or a lower turnover number). In somfl in(cid:173)
`stances, the purified enzyme is fully active, but it may lose its
`ability to be regulated-for example, if the regulatory domain
`of M loses its affinity for a regulatory metabolite. Under such
`circumstances, immobilized lig!llldS with lower affinity for M
`must be tried. Among the remedies that can be used to improve
`
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`74
`
`AFFINITY CHROMATOGRAPHY
`
`Hydrolysis
`
`0
`II
`O-C-NH 3 carbonate (inert)
`
`rea;:~r- ¼ ~H ~ Linear
`'t
`
`lnterchain
`
`~
`~ O-C=N -----<- - - - - o-C - O
`Cyanate ester
`lnterchain
`(very reactive)
`rearrange-
`ment
`~ - - ---
`
`o,
`O/C=NH
`
`im idocarbonate
`(slightly reactive)
`
`Cyclic
`imidocarbonate
`(slightly reactive)
`
`CNBr
`
`~ OH
`~OH
`
`e-NH2
`
`e-NH 2
`
`~
`'
`} o-C - NH-Ligand
`
`O-C-NH- Ligand 't o,
`~ NH
`
`II
`
`.
`o,,C= NH- Ligand
`
`Figure .2. The mechanism of the
`CNBr activation ofagarose.
`(Modified from Ref. 3.)
`
`lsourea
`derivate
`
`N-substituted
`i mi docarbonate
`
`N-substituted
`carbamate
`
`the elution step, one should note the possibility of binding the
`ligand to the matrix by means of an easily cleavable form (cid:173)
`for example, through an ester bond (9,10), which can be read(cid:173)
`ily hydrolyzed with a mild base; through a link that includes
`v:icinal hydroxyl groups, which can be readily cleaved with pe(cid:173)
`riodate; or through diazo bonds, which can be readily reduced
`with dithionates (11). It should be remembered, however, that
`such columns are of limited value, because they can be used
`only once. Electrophoresis has also been used for elution (12).
`Because proteins are charged, they will detach from the col(cid:173)
`umn and migrate toward the appropriate electrode, if the col(cid:173)
`umn with the adsorbed M is exposed to a strong enough electric
`field . This .mild method of elution was successfully applied with
`high yields in immunoaffinity chromatography and in some AC
`systems.
`
`INTERPOSING AN "ARM" BETWEEN THE LIGAND AND THE
`MATRIX BACKBONE
`
`While developing the basic principles of AC, it was observed
`that the purification of M is often improved by interposing
`a hydrocarbon chain (an "arm" or a "spacer") between L and
`the matrix ba.ckbone (l ). It was presumed that s uch an arm
`relieves the steric restrictions imposed by the backbone on
`the ligand, thereby increasing its flexibility and its avail(cid:173)
`ability to the protein (13). Such arms were found to improve
`significantly the extraction of proteins and the efficat,-y of
`the purification by AC. Initially, it was assumed that such
`hydrocarbon arms do not alter the inert nature of the matrix,
`a condition that obviously has to be ensured to preserve an
`active-site mediated adsorption of the extracted protein. This
`assumption seemed reasonable at the time because it had just
`been shown that at least some water-soluble proteins are quite
`well described as "an oil drop with a polar coat" (14), implying
`that the surface of water-soluble proteins is polar and thus not
`
`attracted to lipophilic "baits." We now know that such arms, in
`and of themselves, may bind proteins. In fact, tbjs observation
`led to the discovery of hydrophobic chromatography.
`
`THE LIMITATIONS OF BIOSPECIFICITY-INTERACTIONS
`THAT ARE NOT ACTIVE SITE-MEDIATED
`
`Proteins and their physiological ligands are multifunctional
`molecules whose functions involve a variety of physical in(cid:173)
`teractions: hydrophobic, electrostatic, ion-dipole, and so on.
`Therefore, it is reasonable to assume that a protein might
`interact with a column coated with a ligand (very often an(cid:173)
`chored to the beads at a local concentration much higher than
`its concentration in vivo) not only by means of its active site.
`While it is sometimes possible to minimize these nonspecific
`interactions, it is not always possible to avoid such interfering
`effects, because they may be an intrinsic property of the
`system. For example, if ATP is linked to a matrix through its
`amino group or its ribose moiety; the column thus obtained
`may retain an enzyme having a biospecific site for ATP; but at
`the same time, this very column would be negatively charged
`due to its triphosphate groups, and it would have hydrophobic
`loci due to its adenine residues. Other proteins, in addition to
`the desired one, may therefore "regard" the column material
`as an ion exchanger by virtue of its triphosphate groups, or
`as a hydrophobic column by virtue of its adenin.e moieties. The
`efficiency of resolution will then depend on the magnitude of
`the affinity produced by charge-charge or hydrophobic inter(cid:173)
`actions, as compared to the affinity between the active site of
`the desired macromolecule and its immobilized substrate or
`effector analogue. With columns of macromolecular ligands
`(eg, enzyme subunits, antibodies, lectins), the probability of
`encountering such built-in interfering effects is considerably
`higher, because their immobilization usually involves different
`
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`anchoring points. This leads to a heterogeneous presentation
`of the various regions of the ligand macromolecule. In some of
`these presentations, the biospecific active site is available for
`interaction, while in other presentations the active site itself
`is inaccessible or sterically hindered. Hydrophobic patches
`in uch ligands may be available for interaction not only in
`the bio pecifically functional presentation, but also in other
`presentations. In fact, the tendency of a lectin such as con(cid:173)
`canavalin A to adsorb onto hydrophobic substances, in addition
`to its binding to sugars of the mannosyl configuration, was
`observed in several laboratories.
`
`THE RELATIVITY OF BIOLOGICAL RECOGNITION: DIFFERENT
`PROTEINS MAY SHARE A TASTE FOR A BIORECOGNITION
`ELEMENTS
`
`The occurrence of common biorecognition sites in different
`enzymes is obvious when they are functionally similar, acting
`on the same substrate (eg, ATP), or utilizing the same cofactor
`(eg, NAD ). This actually forms the basis for general ligand(cid:173)
`affinity chromatography (15). However, common biorecog(cid:173)
`nition elements may also be found with proteins having no
`apparent functional similarity. For example, the free catalytic
`subunit of cAMP-dependent protein kinase (protein kinase
`A) is preferentially retarded on immobilized soybean trypsin
`inhibitor (16). Though initially unexpected, this is actually
`not surprising; in spite of the fact that trypsin and this
`kinase catalyze two different chemical reactions (hydrolysis
`of peptide bonds versus a phosphotransferase reaction),
`these two enzymes do have similar biorecognition elements
`(or subsites) at their active site: trypsin cleaves peptide bonds
`adjacent to positively charged amino acid residues (arginine
`and lysine), while cAMP-dependent protein kinase phos(cid:173)
`phorylates serine residues that are vicinal (in the sequence
`of amino acids) to the same positively charged arginine and
`lysine residues (17- 20). Similarly, it was shown (21) that
`TLCK (a -N-tosyl-L-lysine chloromethyl ketone), an affinity
`labeling reagent originally designed for labeling the active
`site of trypsin, specifically attacks a thiol group at the active
`site of the catalytic subunit of cAMP-dependent protein kinase.
`It seems, therefore, that the retardation of the free catalytic
`subunit on the immobilized inhibitor is due (at least in part) to
`an affinity between the inhibitor and recognition subsites at
`the active site of the enzyme.
`
`BIBLIOGRAPHY
`
`1. P. Cuatrecasas, M. Wilchek, and C. 8 . Anfinsen (1968) Proc. Natl.
`Acad. Sci. USA 61 , 636.
`2. P . Cuatrecasas and C. B. Anfin en (1971) Annu. Rev. Biochem.
`40,259.
`3. M. Wilchek, T. Miron, and J. Kohn ( 1984) Methods Enzymol. 104,
`3.
`4. R. Axen, J . Porath, and S. Ernback (1967) Nature (London) 214,
`1302.
`5. J . Kohn and M. Wilchek (1981) Anal. Biochem. 115, 375.
`6. S. 8 . Mohan and A. Lydd.iatt (1997) Jn Affinity Separations
`(P. Matejtschuk, ed.), IRL Press, Oxford University Press, New
`York, p. 1.
`7. S. Shaltiel, J . L. Hedrick, and E. H. Fischer (]966) Biochemistry
`5, 2108.
`
`AFFINITY ELECTROPHORESIS
`
`75
`
`8. J . L. Hedrick, S. Shalliel, and E. H. Fischer (1969) Biochemistry
`8, 2422.
`9. R. J . Brown, N. E. Swaisgood, and H. R. Horton (1979) Biochem(cid:173)
`istry 18, 4901.
`10. P. Singh, S. D. Lewis, and J . A. Shafer (1979) Arch. Biochem.
`Biophys. 193,284.
`11. P . Singh, S. D. Lewis, and J. A. Shafer (1980) Arch. Biochem.
`Biophys. 203, 776.
`12. M. R. Morgan, P . J . Brown, M. J . Lieia nd, and P. D. Ocan (1978)
`FEBS Lett. 87, 239.
`13. P . Cuatrecasas (1970) J . Biol. Chem. 245, 3059.
`14. D. C. Phillips (1966) Sci. Am. November, 78.
`15. K. Mosbach (1978) Adu. Enzymol. 46, 205.
`16. E. Alhanaty, N. Bashan, S. Moses, and S. Shaltiel (1979) Eur. J.
`Biochem. 101, 283.
`17. H . G. Nimmo and P. Cohen (1977) Adu. Cyclic Nucl. Res. 8, 145.
`18. 0 . Zetterquist et al. (1976) Biochem. Biophys. Res. Comun. 70,
`696.
`19. B. E. Kemp, E. Benjamin, and E. G. Krebs (1976) Proc. Natl.
`Acad. Sci. USA 73, 1038.
`20. P. Daile, P . R. Carnegie, and J . D. Young (1975) Nature 257, 416.
`21. A. Kupfer, V. Gani, J . S. Jimenez, and S. Shaltiel (1979) Proc.
`Natl. Acad. Sci. USA 76, 3073.
`
`AFFINITY ELECTROPHORESIS
`
`A. CHRAMBACH
`
`By analogy to affinity chromatography, it is possible to in(cid:173)
`troduce specific ligands for a macromolecule into the gels of
`gel electrophoresis and to measure the specific retardation
`of the macromolecule due to its interaction with such a reagent.
`The advantage of such affinity methods lies in the augmented
`resolving power conferred by the specificity of the binding
`interaction.
`The procedures used to introduce affinity reagents into
`gels have varied. In cross electrophoresis, a ligand with
`a net charge opposite to the species of interest migrates
`electrophoretically into the gel in the opposite direction.
`Alternatively, uncharged ligands can simply be added to the
`gelation mixture. Macromolecular substrates within a gel may
`serve as immobilized affinity reagents, either by themselves
`or as carriers of covalently attached affinity groups. The
`magnitude of the electrophoretic retardation depends on the
`concentration of the affinity reagent in the gel; quantitative
`determination of this relationship makes it possible to es(cid:173)
`timate the apparent association constant for binding of the
`ligand to the sample. Further information concerning the
`interaction can be gained from affinity electrophoresis by
`variation of the buffer composition (eg, the addition of metal
`ions to the buffer), the pH, or the temperature.
`
`Suggestions for Further Reading
`T. C. Bog-Hansen and J . J . Hau (1981) Glycoproteins and glycopep(cid:173)
`tides (affinity electrophoresis). In Electrophoresis: A Survey of
`Techniques and Applications, Vol. 18B (Z. Deyl, A. Chrambach,
`F. M. Everaerts, and Z. Prusik, eds.), Elsevier, Amsterdam,
`pp. 219-252.
`T. C. Bog-Hansen and K. Takeo, eds. (1989) Symposium on affinity
`electrophoresis. Electrophoresis 10, 811 - 870.
`
`6 of 46
`
`Fresenius Kabi
`Exhibit 1008
`
`

`

`192
`
`ARGININE (ARG, R)
`
`markers, on average, is 200 kb per cM. However, from the
`physical map construction of chromosome 4, it was noticed
`that recombination hot spots (30 to 50 kb/cM) and low spots
`(~ 550 kb/cM) do occur (13). Many tools are available to map a
`mutation, for example, recessive visible markers, codominant
`embryo-lethal markers, dominant selectable markers on the
`located T-DNA and Ac/Ds insertions, restriction fragment
`length polymorphism (RFLP)-derived and PCR-based
`molecular markers, such as microsatellites. Several RFLP,
`rapid-amplified polymorphic DNA, or amplified fragment
`length polymorphism (AFLP) molecular marker maps have
`been constructed, based on different mapping populations.
`A combined map, made by statistical integration, gives an
`approximate position and order of the markers. Recombinant
`inbred (RI) lines, derived from a cross between Columbia and
`Landsberg erecta (14), have been used to locate more than
`750 molecular markers unambiguously. Genes are mapped
`by RFLP segregation analysis by using RI lines or by matrix(cid:173)
`based PCR analysis of pooled yeast artificial chromosome
`clones (YACs). The physical map consists of contigs of DNA
`clones that are correlated with the mapped markers. Cur(cid:173)
`rently, YAC, bacterial artificial chromosome (BAC), and
`phage Pl artificial chromosome (PAC) contig. maps are
`available that cover almost the entire genome. Genetic and
`physical maps are updated through AtDB.
`
`SCIENTIFIC ADVANCES AND APPLICATIONS
`
`The molecular-genetic approach in Arabidopsis research has
`led to major breakthroughs in plant developmental biology.
`Tremendous progress has been made in understanding the
`molecular control ofmeristem identity during flower initiation
`and flower organ formation, embryo development and pattern
`formation during embryogenesis, root development, epidermal
`cell fate specification in root hair and trichome formation,
`and cell determination in the vegetative meristem. Genes
`have been identified that are involved in hormone perception,
`biosynthesis, and signal transduction. The first hormone
`receptor for plants has been characterized in Arabidopsis
`(15). Much of the molecular insights into light perception
`and signal transduction, cell cycle regulation, and disease
`resistance in higher plants comes from studies in Arabidopsis
`(16-18).
`TheArabidopsis genes and mutants are resources exploited
`either to isolate orthologs from other species and to test their
`functional conservation (19) or to be used straight away for
`the genetic modification of even distantly related crop plants
`(20). The molecular markers within contigs in Arabidopsis
`have been used for comparative mapping with Brassica spp.
`Colinearity in 5- to 10-cM regions has been demonstrated be(cid:173)
`tween the Arabidopsis genome and that of Brassica nigra (21).
`This implies that information and markers obtained from the
`physical mapping in Arabidopsis can be applied to syntenic
`genomic regions in mustard crops to analyze important traits
`in breeding programs.
`
`BIBLIOGRAPHY
`
`1. F. Laibach (1943) Bot. Archiu 44, 439-455.
`2. M. D. Bennett and J.B. Smith (1976) Philos. Trans. R. Soc. Lond.
`B Biol. Sci. 274, 2.27-274.
`
`3. G. P. Redei (1975)Ann. Reu. Genet. 9, 111- 127.
`4. M. Koomneef et al. (1983) J. Hered. 74, 265-272.
`5. L. S. Leutwiler, B. R. Hough-Evans, and E. M. Meyerowitz (1984)
`Mol. Gen. Genet. 194, 15-23.
`6. D. Valvekens, M. Van Montagu, and M. Van Lijsebettens (1988)
`Proc. Natl. Acad. Sci. USA 86, 5536-5540.
`7. K. A. Feldmann (1991) Plant J . 1, 71-82.
`8. National Science Foundation (1990) A long-range plan for the
`multinational coordinated Arabidopsis thaliana genome research
`project (NSF 90-80), National Science Foundation, Washington,
`DC. (published annually).
`9. R. E. Pruitt and E. M. Meyerowitz (1986) J. Mol. Biol. 187, 169-
`183.
`10. T. Newman et al. (1994) Plant Physiol. 106, 1241-1255.
`11. R. Cooke et al. (1996) Plant J . 9, 101-124.
`12. M. Bevan et al. (1998) Nature (London) 391, 485-488.
`13. R. Schmidt et al. (1995) Science 270, 480-483.
`14. C. Lister and C. Dean (1993) Plant J. 4, 745-750.
`15. G. E. Schaller and A. B. Bleecker (1995) Science 270, 1809-1811.
`16. J. L. Dang! (1995) Cell 80, 363-366.
`17. C. Lin et al. (1995) Science 269, 968-970.
`18. A. Hemerly et al. (1992) Proc. Natl. Acad. Sci. USA 89, 3295-
`3299.
`19. V. F . Irish and Y. T. Yamamoto (1995) Plant Cell 1, 1635-1644.
`20. D. Weigel and 0 . Nilsson (1995) Nature 877, 495-500.
`21. U. Lagercrantz, J. Putterill, G. Coupland, and D. Lydiate (1996)
`Plant J . 9, 13-20.
`
`Suggestions for Further Reading
`M. Anderson and J. Roberts (1998) Arabidopsis (Annual Plant Re(cid:173)
`views, Vol. 1), Sheffield Academic Press, Sheffield, UK.
`J. Bowman (1994) Arabidopsis: an Atlas of Morplwlogy and Develop(cid:173)
`ment, Springer-Verlag, New York.
`C. Koncz, N .-H. Chua and J . Schell (1992) Methods in Arabidopsis
`Research, World Scientific, Singapore.
`J. M. Martinez-Zapater and J. Salinas (1998) Arabidopsis Protocols
`(Methods in Molecular Biology, Vol. 82) The Humana Press, To(cid:173)
`towa, NJ.
`E. M. Meyerowitz and C. R. Somerville (1994) Arabidopsis (Cold
`Spring Harbor Monograph Series, Vol. 27), Cold Spring Harbor
`Laboratory Press, Cold Spring Harbor, NY.
`
`ARGININE (ARG, R)
`
`T. E. CREIGHTON
`
`The amino acid arginine is incorporated into the nascent
`polypeptide chain during protein biosynthesis in re(cid:173)
`sponse to six codons-CGU, CGC, CGA, CGG, AGA, and
`AGG-and represents approximately 5. 7% of the residues
`of the proteins that have been characterized. The arginyl
`residue incorporated has a mass of 156.19 Da, a van der
`Waals volume of 148 A3
`, and an accessible surface area
`of 241 A2
`• Arg residues have average conservation during
`divergent evolution; they are interchanged most fre(cid:173)
`quently in homologous proteins with lysine, the other basic
`residue.
`
`7 of 46
`
`Fresenius Kabi
`Exhibit 1008
`
`

`

`The Arg side chain consists of three nonpolar methylene
`groups and the strongly basic <5-guanido group:
`
`With a pK0 value usually of about 12, the guanido group
`is ionized over the entire pH range in which proteins exist
`naturally. The ionized guanido group is planar as a result of
`resonance:
`
`1l
`
`11
`
`and the positive charge is effectively distributed over the en(cid:173)
`tire group. 1n the protonated form, the guanido group is unre(cid:173)
`active, and only very small fractions of the nonionized form are
`present at physiological pH values. The guanido groups of Arg
`residues are almost invariably at the surfaces of native protein
`structures, and virtually no Arg residues are fully buried, but
`the nonpolar part of the side chain, and the adjoining polypep(cid:173)
`tide backbone, are frequently buried within the interior. Arg
`residues favor the alpha-helical conformation in model pep(cid:173)
`tides and also occur most frequently in that secondary struc(cid:173)
`ture in folded protein structures.
`Proteinases frequently cleave polypeptide chains adjacent
`to Arg residues, as in the processing of pro-hormones, such as
`pro-insulin, at pairs of basic residues.
`The guanido group can form heterocyclic condensation
`products with 1,2- and 1,3-dicarbonyl compounds, such as
`phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione:
`
`ASCARIS
`
`193
`
`Arg
`
`cyclohexanedione
`
`1l
`
`These reactions occur readily because the distance between the
`two carbonyl groups of the reagents closely matches that be(cid:173)
`tween the two unsubstituted nitrogen atoms of the guanido
`group. The adduct formed can be stabilized further by the pres(cid:173)
`ence of borate, which complexes with the adjacent hydroxyl
`groups.
`The guanido group can be cleaved by hydrazine (H:zNNH2)
`to produce the side chain of ornithine:
`
`Arg l H2N-NH2
`
`-CH2-CH2-CH2-NH2
`
`Orn
`
`This reaction is, however, often accompanied by cleavage of the
`polypeptide backbone.
`
`SUlfgestio,.. for Further Reading
`
`E. L. Smith (1977) Reversible blocking at arginine by cyclohexane(cid:173)
`dione, Meth. Enzyrrwl. 47, 156-161.
`A Honegger et al. ( 1981) Chemical modification of peptides by hy(cid:173)
`drazine, Biochem. J. 199, 53-59.
`R. B