[56]
PROTEINASE
INHIBITORS
FROM
PLANT
SOURCES
695
acetone-acetic acid-5% ammonia-water (35: 25:15 : 15: 10) on cellulose (Avicel). Biological Activity. Phosphoramidon inhibits thermolysin and the related enzyme; very specifically. The intravenous injection of 1.0 g/kg does not cause death in mice. Kinetic Properties2 Inhibition of thermolysin by phosphoramidon is competitive with substrate; K~ for earbobenzoxy-glyeyl-L-leucine amide is 28 nM. It is of interest that phosphoryl-L-leucyl-L-tryptophan (a mild hydrolysis product of phosphoramidon) shows a stronger inhibitory activity: K~ = 2.0 riM.
[56] P r o t e i n a s e I n h i b i t o r s f r o m P l a n t S o u r c e s B y YEHUDITH BIRK
Distribution and Occurrence Proteinlike proteinase inhibitors are widely distributed in the plant kingdom. Most of the inhibitors are present in the seeds of the various plants, but they are not necessarily restricted to this part of the plant. Certain storage organs, such as seeds from the Leguminosae family and tubers from the Solanaeeae family, are excellent sources of proteinase inhibitors. The inhibitors are diverse in number and in specificity toward various proteolytie enzymes. Several different kinds of inhibitors can be present in a single tissue--for example, barley grains, soybeans, and potato tuber. The multiplicity of plant proteinase inhibitors may partly be ascribed to the self- and mixed-association of a few monomers in each plant as well as to partial proteolysis of the inhibitors during purification, especially when the inhibitors are purified by affinity chromatography. The frequent presence of several proteinase inhibitors in the same source tissue, and the finding that the same inhibitor often inhibits more than one enzyme, are partly responsible for the difficulty in establishing their nomenclature. The physiological significance of plant proteinase inhibitors has been questioned for a long time. The fact that certain seeds, such as soybeans and wheat grains, contain inhibitors of growth and of larval gut proteases of the insects Triboliurn and Tenebrio, 1 suggest the possibility that these inhibitors may have evolved as a defense mechanism against predatory 1y. Birk, Proteinase Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 355. Springer-Verlag, Berlin and New York, 1974.
696
NATURALLY OCCURRING PROTEASE INHIBITORS
[56]
insects. 2 This working hypothesis is further supported by the finding t h a t wounding of the leaves of potato or tomato plants by insects induces a rapid accumulation of chymotrypsin inhibitor I2 This process is associated with the release and translocation of a hormone t h a t induces rapid accumulation of proteinase inhibitors, 4 and it thus demonstrates t h a t insect behavior can influence the protein composition of plant leaves. Characterization The molecular weights of plant inhibitors are mainly in the range from 3000 to 25,000. M a n y of them have been isolated in pure form and characterized with respect to amino acid composition, partial or full amino acid sequence, chemistry of the reactive sites, and the nature of the complexes formed with the respective proteinases. The inhibitors are frequently multiheaded as a consequence of gene elongation (via gene duplication or multiplication); this feature is being used for the study of evolution of specificity proteinase inhibitors2 I t is generally assumed t h a t inhibition of several enzymes by the same inhibitor is done either at separate nonoverlapping reactive sites or at separate but overlapping reactive sites, or at the same reactive site. Several mechanisms have been proposed ~,7 for enzyme-inhibitor interactions.
General Methods of Assay I n h i b i t o r y activity and inhibitor concentration measurements are based on formation of enzyme-inhibitor complex and determine the decrease in the enzymic hydrolysis of natural or synthetic substrates. Inhibitor concentration can be determined in crude extracts provided the association constant of enzyme-inhibitor is high and the exact concentration of the active enzyme is known. For determination of specific inhibitory activity, assays should be performed at 50% inhibition of the enzyme, provided t h a t inhibition is stoichiometric in this range. SufS. w. Applebaum and Y. Birk, in "Insect and Mite Nutrition" (J. G. Rodriguez, ed.), p. 629. North-Holland Publ., Amsterdam, 1972. a T. R. Green and C. A. Ryan, Science 175, 776 (1972). * T. R. Green and C. A. Ryan, Plant Physiol. 51, 19 (1972). M. Laskowski, Jr., I. Kato, T. R. Leary, J. Schrode, and R. Sealock, Proteinase Inhibitors, Proc. Int. Res: ConJ., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 597. Springer-Verlag, Berlin and New York, 1974. e W. R. Finkenstadt, M. A. Hamid, J. A. Mattis, J. Schrode, R. Sealock, D. Wang, and M. Laskowski, Jr., Proteinase Inhibitors, Proc. Int. Res. Conj., 2nd, (Bayer Symp. V), Grosse Ledder, 1973, p. 389. Springer-Verlag, Berlin and New York, 1974. H. Ako, R. J. Foster, and C. A. Ryan, Biochemistry 13, 132 (1974).
[57]
PROTEINASE INHIBITORS FROM LEGUME SEEDS
697
ficient preincubation of enzyme and inhibitor should be allowed to reach inhibition equilibrium. Chapters [56]-[63] deal with the most characterized inhibitors from plant sources. As stated already by KasselP in this series, it has not been possible to express the units of inhibiting activity in a uniform manner; the assays and units are those used by the individual investigators whose isolation procedures are described. The chapter by Kassell, 8 the reviews by Liener and K a k a d e ° and by Laskowski, Jr. and Sealock, 1° and the Proceedings of the First and Second International Research Conferences on Proteinase Inhibitors 11,~ should be consulted for earlier as well as for further information. Acknowledgment The author is grateful to Dr. R. Hofstein for her most valuable assistance in preparing Chapters [56]-[63]. 8B. Kassell, this series Vol. 19 [66]. ' I . E. Liener and M. L. Kakade, in "Toxic Constituents of Plant Foodstuffs" (I. E. Liener, ed.), p. 8. Academic Press, New York, 1969. 10M. Laskowski, Jr., and R. W. Sealock, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 3, p. 375. Academic Press, New York, 1971. 11Proteinase Inhibitors Proc. First Int. Res. Con]. 1st, Munich, 1970, de Gruyter, Berlin, 1971. 12Proteinase Inhibitors, Proc., Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973. Springer-Verlag, Berlin and New York, 1974.
[57] Proteinase
Inhibitors
from
Legume Seeds
B y YEHUDITH BIRK
The presence of proteinlike trypsin inhibitors in legume seeds is by now a well established fact. Different legume seeds contain one or more inhibitors. Most of them have a molecular weight of about 8000 with a high concentration of cystine ( ~ 2 0 % ) and no free SH groups. The abundance of S - - S bonds accounts for the considerable resistance to overall denaturation and to proteolytic digestion. Establishment of the amino acid sequence of the B o w m a n - B i r k soybean inhibitor 1 and of the lima bean inhibitor I V 2 shows the existence of two homologous regions in these proteins. The region located in the first half of the molecule S. Odani and T. Ikenaka, J. Biochem. (Tokyo) 71,839 (1972). : F. C. Stevens, S. Wuerz, and J. Krahn, Proteinase Inhibitors, Proc. Int. Res. Con/., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 344. Springer-Verlag, Berlin and New York, 1974.
PROTEINASE
INHIBITORS
FROM
PLANT
SOURCES
695
acetone-acetic acid-5% ammonia-water (35: 25:15 : 15: 10) on cellulose (Avicel). Biological Activity. Phosphoramidon inhibits thermolysin and the related enzyme; very specifically. The intravenous injection of 1.0 g/kg does not cause death in mice. Kinetic Properties2 Inhibition of thermolysin by phosphoramidon is competitive with substrate; K~ for earbobenzoxy-glyeyl-L-leucine amide is 28 nM. It is of interest that phosphoryl-L-leucyl-L-tryptophan (a mild hydrolysis product of phosphoramidon) shows a stronger inhibitory activity: K~ = 2.0 riM.
[56] P r o t e i n a s e I n h i b i t o r s f r o m P l a n t S o u r c e s B y YEHUDITH BIRK
Distribution and Occurrence Proteinlike proteinase inhibitors are widely distributed in the plant kingdom. Most of the inhibitors are present in the seeds of the various plants, but they are not necessarily restricted to this part of the plant. Certain storage organs, such as seeds from the Leguminosae family and tubers from the Solanaeeae family, are excellent sources of proteinase inhibitors. The inhibitors are diverse in number and in specificity toward various proteolytie enzymes. Several different kinds of inhibitors can be present in a single tissue--for example, barley grains, soybeans, and potato tuber. The multiplicity of plant proteinase inhibitors may partly be ascribed to the self- and mixed-association of a few monomers in each plant as well as to partial proteolysis of the inhibitors during purification, especially when the inhibitors are purified by affinity chromatography. The frequent presence of several proteinase inhibitors in the same source tissue, and the finding that the same inhibitor often inhibits more than one enzyme, are partly responsible for the difficulty in establishing their nomenclature. The physiological significance of plant proteinase inhibitors has been questioned for a long time. The fact that certain seeds, such as soybeans and wheat grains, contain inhibitors of growth and of larval gut proteases of the insects Triboliurn and Tenebrio, 1 suggest the possibility that these inhibitors may have evolved as a defense mechanism against predatory 1y. Birk, Proteinase Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 355. Springer-Verlag, Berlin and New York, 1974.
696
NATURALLY OCCURRING PROTEASE INHIBITORS
[56]
insects. 2 This working hypothesis is further supported by the finding t h a t wounding of the leaves of potato or tomato plants by insects induces a rapid accumulation of chymotrypsin inhibitor I2 This process is associated with the release and translocation of a hormone t h a t induces rapid accumulation of proteinase inhibitors, 4 and it thus demonstrates t h a t insect behavior can influence the protein composition of plant leaves. Characterization The molecular weights of plant inhibitors are mainly in the range from 3000 to 25,000. M a n y of them have been isolated in pure form and characterized with respect to amino acid composition, partial or full amino acid sequence, chemistry of the reactive sites, and the nature of the complexes formed with the respective proteinases. The inhibitors are frequently multiheaded as a consequence of gene elongation (via gene duplication or multiplication); this feature is being used for the study of evolution of specificity proteinase inhibitors2 I t is generally assumed t h a t inhibition of several enzymes by the same inhibitor is done either at separate nonoverlapping reactive sites or at separate but overlapping reactive sites, or at the same reactive site. Several mechanisms have been proposed ~,7 for enzyme-inhibitor interactions.
General Methods of Assay I n h i b i t o r y activity and inhibitor concentration measurements are based on formation of enzyme-inhibitor complex and determine the decrease in the enzymic hydrolysis of natural or synthetic substrates. Inhibitor concentration can be determined in crude extracts provided the association constant of enzyme-inhibitor is high and the exact concentration of the active enzyme is known. For determination of specific inhibitory activity, assays should be performed at 50% inhibition of the enzyme, provided t h a t inhibition is stoichiometric in this range. SufS. w. Applebaum and Y. Birk, in "Insect and Mite Nutrition" (J. G. Rodriguez, ed.), p. 629. North-Holland Publ., Amsterdam, 1972. a T. R. Green and C. A. Ryan, Science 175, 776 (1972). * T. R. Green and C. A. Ryan, Plant Physiol. 51, 19 (1972). M. Laskowski, Jr., I. Kato, T. R. Leary, J. Schrode, and R. Sealock, Proteinase Inhibitors, Proc. Int. Res: ConJ., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 597. Springer-Verlag, Berlin and New York, 1974. e W. R. Finkenstadt, M. A. Hamid, J. A. Mattis, J. Schrode, R. Sealock, D. Wang, and M. Laskowski, Jr., Proteinase Inhibitors, Proc. Int. Res. Conj., 2nd, (Bayer Symp. V), Grosse Ledder, 1973, p. 389. Springer-Verlag, Berlin and New York, 1974. H. Ako, R. J. Foster, and C. A. Ryan, Biochemistry 13, 132 (1974).
[57]
PROTEINASE INHIBITORS FROM LEGUME SEEDS
697
ficient preincubation of enzyme and inhibitor should be allowed to reach inhibition equilibrium. Chapters [56]-[63] deal with the most characterized inhibitors from plant sources. As stated already by KasselP in this series, it has not been possible to express the units of inhibiting activity in a uniform manner; the assays and units are those used by the individual investigators whose isolation procedures are described. The chapter by Kassell, 8 the reviews by Liener and K a k a d e ° and by Laskowski, Jr. and Sealock, 1° and the Proceedings of the First and Second International Research Conferences on Proteinase Inhibitors 11,~ should be consulted for earlier as well as for further information. Acknowledgment The author is grateful to Dr. R. Hofstein for her most valuable assistance in preparing Chapters [56]-[63]. 8B. Kassell, this series Vol. 19 [66]. ' I . E. Liener and M. L. Kakade, in "Toxic Constituents of Plant Foodstuffs" (I. E. Liener, ed.), p. 8. Academic Press, New York, 1969. 10M. Laskowski, Jr., and R. W. Sealock, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 3, p. 375. Academic Press, New York, 1971. 11Proteinase Inhibitors Proc. First Int. Res. Con]. 1st, Munich, 1970, de Gruyter, Berlin, 1971. 12Proteinase Inhibitors, Proc., Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973. Springer-Verlag, Berlin and New York, 1974.
[57] Proteinase
Inhibitors
from
Legume Seeds
B y YEHUDITH BIRK
The presence of proteinlike trypsin inhibitors in legume seeds is by now a well established fact. Different legume seeds contain one or more inhibitors. Most of them have a molecular weight of about 8000 with a high concentration of cystine ( ~ 2 0 % ) and no free SH groups. The abundance of S - - S bonds accounts for the considerable resistance to overall denaturation and to proteolytic digestion. Establishment of the amino acid sequence of the B o w m a n - B i r k soybean inhibitor 1 and of the lima bean inhibitor I V 2 shows the existence of two homologous regions in these proteins. The region located in the first half of the molecule S. Odani and T. Ikenaka, J. Biochem. (Tokyo) 71,839 (1972). : F. C. Stevens, S. Wuerz, and J. Krahn, Proteinase Inhibitors, Proc. Int. Res. Con/., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 344. Springer-Verlag, Berlin and New York, 1974.
