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Plant Physiol. (1978) 61, 501-505 Proteases of Senescing Oat Leaves II. REACTION TO SUBSTRATES AND INHIBITORS' Received for publication March 23, 1977 and in revised form November 2, 1977 ROLF H. DRIVDAHL2 AND KENNETH V. THIMANN3 Thimann Laboratories, University of California, Santa Cruz, California 95064 ABSTRACT MATERIALS AND METHODS Two proteases isolated from senescent oat (Avens saiva) leaves have Plant Material. Seeds of A. sativa cv. Victory, obtained from been subjected to further study. One of these, an acid protease active at the USDA, were husked, soaked, and grown in Vermiculite as pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by described previously (23). After 8 days of growth in light the intact iodoacetamide (lAc). The other, active at pH 6.6, is inhibited by both plants were left in darkness for 2 to 3 days, in order to maximize PMSF and IAc. These results, together with previously reported evidence protease yields. that mercaptoethanol stimulates the activity of only the neutral protease, Protease Extraction and Purification. Procedures for homogeare taken to indicate that the acid protease is probably of the serine type, nization of the tissue and purification of the extracts have been whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are described (9). For the experiments presented here, the final step in Inhibited by irradiation in the presence of rose bengal, a selective histidine purification, involving chromatography on DEAE-Sephadex, was modification reagent. The acid protease was completely unaffected by omitted. Material eluted from a hemoglobin-Sepharose affinity chelators, but data on the neutral protease were equivocal. column was dialyzed against 10 mm sodium phosphate buffer (pH All protein substrates tested were attacked by both enzymes, though at 6) at 0 C, and either used directly or concentrated by ultrafiltration strkingly different rates. Characterization of the digestion products, with in an Amicon cell equipped with a PM-10 membrane. As shown denatured hemogiobin as substrate, indicated that the acidic enzyme is an in the preceding paper (9), the enzymes are highly purified at this endoprotease, while the neutral one is an exoprotease. Evidence is pre- point, and omission of the DEAE step avoids the large loss of sented that these proteases undergo autolysis in vitro. activity normally encountered at the final stage; the primary disadvantage is that they are not separated from each other, since this was accomplished by the ion exchanger. However, their effects were easily differentiated by working at the respective pH optima. Protease Assay. Except where indicated, both proteases were assayed with denatured hemoglobin as substrate; details of the Several studies in recent years have indicated a major role for incubation procedure and analysis of the reaction products were proteases in the senescence of leaves (1, 13, 17, 25). In at least one described in the previous report (9). One unit is defined as that case the observed increases in proteolytic activity have correlated amount of enzyme which liberates I ,umol of leucine equivalents with the liberation of a-amino nitrogen which is such a character- in 1 hr at 50 C. Use of this high temperature, which was shown istic concomitant of the senescence process. In senescing leaves of previously (9) to be the optimum for short incubations, gave Avena sativa, previous work from this laboratory has shown the results qualitatively similar to those obtained at lower temperapresence of at least two proteases (17). One of these, active at acid tures, while permitting the use of smaller enzyme quantities and pH values, showed marked increases in activity during the brief shorter reaction times. The optimum assay temperature for the experimental senescence period, while the other, active in the acid protease of wheat seedling leaves has been reported as 52 C; neutral range, increased more slowly. Extensive purification and the reaction rate at 50 C was linear for at least 90 min (11). a preliminary characterization of these two proteases have now Inhibitor Studies. pCMB' was dissolved in 20 mm sodium been reported (9). They have virtually identical mol wt, as deter- acetate buffer (pH 5), and equal volumes of inhibitor and enzyme mined by gel filtration, show unexpectedly high temperature stock solution were incubated for 30 min at 30 C. The exact optima, close to 50 C, and both exert their activity over broad pH concentration of the pCMB stock solution was determined specranges. The neutral enzyme required the presence of a sulfhydryl trophotometrically, using a molar extinction coefficient of 1.69 x compound for maximal activity. 104 M-1 cm-' (6). Protease activity remaining after these and all The present work extends the biochemical study of these two other preincubations with inhibitors was estimated by the standard proteases. Evidence is adduced that the acidic enzyme is probably hemoglobin assay. an endoprotease, having seine at the active site, while the neutral PMSF was dissolved in 100%o ethanol and diluted to 0.1 M with enzyme appears to be an exoprotease and has a sulfhydryl group 20 mm sodium phosphate buffer (pH 7). Concentrations of 10 mm at its active site. Both probably undergo autolysis in vitro just as PMSF in 10%o ethanol were obtainable in this fashion. Equal they probably do in vivo as the leaf senescence progresses (17). volumes of inhibitor and enzyme were mixed and the reaction Given the apparently generalized nature of the protein degrada- allowed to proceed at 30 C for 30 min. The final concentration of tion that occurs during senescence, the presence of proteases of ethanol in all preincubations was thus 5%; controls in 5% ethanol varying activities and specificities in the leaf is not unexpected. without inhibitor indicated negligible effects of this level of ethanol on protease activity. 'Supported in part by National Science Foundation Grant GB 35238 to K. V. T. 2 Present address: 1521 Pine Street, Olympia, Washington 98502. 4 Abbreviations used in this paper: lAc: iodoacetamide; pCMB: parachloromercuribenzoic acid; PMSF: phenylmethylsulfonyl fluoride. 'To whom requests for reprints should be sent. Downloaded from on June 18, 2017 501- Published by www.plantphysiol.org Copyright © 1978 American Society of Plant Biologists. All rights reserved. 502 DRIVDAHL AND THIMANN Reaction with lAc was carried out at pH 7 in 20 mm sodium phosphate buffer; the incubation times were varied, and these are reported with the results. Solutions of rose bengal in phosphate-citrate buffer were mixed with equal volumes of enzyme and irradiated at room temperature with a 450-w spotlight for 5 min. The light was placed 5 cm from the tubes, and a distilled H20 screen was positioned between the tubes and the light to minimize heating. Controls contained rose bengal but were protected from the light by wrapping in aluminum foil; the dye was removed from the mixture by chromatography on Sephadex G-25 (coarse) prior to enzyme assay. Metal ions and metal chelators were dissolved in the appropriate assay buffers, and the enzymes were added in the standard assay proportions previously described (9). The mixtures were then incubated for 30 min at 30 C prior to addition of the substrate. Substrate Specifcity Studies. Gelatin, casein, and BSA were dissolved or suspended in water and simply substituted for hemoglobin in the standard assays. Azocoli (Calbiochem), a powdered insoluble cowhide derivative covalently bound to a red dye, was added in 10-mg amounts to small flasks containing 1 ml of enzyme solution and 4 ml ofthe appropriate assay buffer. Samples were incubated at the optimum temperature (50 C) for both enzymes with sufficiently vigorous shaking to keep the substrate particles in an even suspension. The reaction was similar at 37 C, though naturally slower. Filtration of the mixture through lens paper removed the residual substrate and thus terminated the reaction; the A of the filtrate was read at 520 nm. Chromatography of Reaction Products. To compare the products of proteolysis at pH 4.2 and 6.6, the reactions were run as described above, but with the volumes scaled up by a factor of 10. The reactions were terminated by immersing the tubes in a boiling water bath for 5 min. At pH 4.2 the hemoglobin was not precipitated by boiling, and this necessitated the addition of sufficient 0.4 M Na2HP04 to raise the pH above 6 before boiling. The mixtures were cooled at 0 C for 1 hr and then centrifuged; the resultant clear supernatants were concentrated in an air stream. The concentrated solutions were then applied to a column (1.5 x 95 cm) of Sephadex G-50 (medium) equilibrated with 50 mm sodium phosphate (pH 7.2). From this column 2-ml fractions were collected and the soluble a-amino nitrogen determined by the ninhydrin reaction. Substrate Specificity-Synthetic Substrates. Several synthetic protease substrates were tested with both the acidic and neutral proteases, in hopes of learning more about their peptide bond specificity and of finding a more convenient substrate for kinetic Plant Physiol. Vol. 61, 1978 work. All compounds were obtained from Sigma Chemical Corp. All were assayed spectrophotometrically, making use of the shift in the absorption maximum between the substrate and the product of hydrolysis. Difference spectra maxima employed were as follows: p-tosyl-L-arginine methyl ester, 247 nm; benzoyl-L-arginine ethyl ester, 253 nm; benzoyl-L-tyrosine ethyl ester, 256 nm; benzoyl-L-arginine p-nitroanilide, 410 nm. The substrates were dissolved in dimethylsulfoxide and diluted into appropriate assay buffer, and 1.5 ml of this solution was mixed with 0.5 ml of suitably diluted enzyme stock and incubated at 35 C for I hr. Results were expressed as the change in A of the reaction mix in the 1-hr period. Soybean trypsin inhibitor was from Sigma. Tests for Autolysis in Solution. To study the possible reappearance of small mol wt peaks, samples of the partially purified protease from the affinity chromatography step were applied to a column (1.5 x 90 cm) of Sephadex G-100 in phosphate-citrate buffer (pH 6). Because of their closely similar mol wt, both proteases eluted in a single symmetrical peak. The active fractions were pooled, concentrated, stored for 72 hr at 6 C, and then rechromatographed on the same column. The appearance of new peaks indicated proteolysis. RESULTS Kinetics. In the particular case of proteolytic enzymes, Lineweaver-Burk plots and determinations of Km values can be complicated, since if some protein substrate is introduced as an impurity, then even in the absence of hemoglobin or other added substrate some proteolysis occurs. This is a more serious problem at low substrate concentrations than at saturating levels, for in the latter the relatively enormous amounts of hemoglobin in the assay mixture render any contribution of substrate by the enzyme preparation negligible. This difficulty was overcome by measuring the small amount of hydrolysis occurring in the absence of added hemoglobin and subtracting this from all other values. The resulting linear relationship between 1/S and l/V, obtained for both Avena proteases, is shown in Figure 1, a and b. The approximate Km values from the figure are 0.11% hemoglobin for the acidic protease and 0.067% for the neutral one. Although the Km for the neutral enzyme is lower, the Vmaz is also lower. This effect is more specifically a feature of the substrate than of the enzyme, since experiments reported below, using other protein substrates, indicate a higher V.. for the neutral protease. The acidic protease of wheat leaves yields a value of 0.026% (I 1). It was somewhat surprising to discover that the acidic protease, 2. I1.9 I 1.0 1.01 0.8[ 0.8~ 0Vo. Km -O.115% 0.6k Heoglobin Km a Q067 % Hemogloin 0.4 0.4 0.2 021 a I 5 I- lS 'S 20 I~~~~~~~~~~~~~~~ 25 30 b F I I 5 10 I 15 1 I 20 25 30 FIG. 1. Lineweaver-Burk plots depicting the relationship between substrate (hemoglobin) concentration and rate of hydrolysis by the A vena proteases at 50 C. a: Neutral protease; b: acidDownloaded protease. from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 61, 1978 PROTEASES OF SENESCING OAT LEAVES Table I Hydrolysis of different proteins by Avena leaf proteases Azocoll was used as a suspension at 10 mg/ml; all other substrates at a concentration of 4%. Assay was performed at 50 C for 90 min. A unit is defined as the amount of enzyme that liberates one imol of amino nitrogen per hr. for all substrates except Azocoll, for which one unit is the amount of enzyme which produces a AOD520 of 0.1 in 1 hr. Substrate 503 The photosensitive dye rose bengal has been shown to be a highly selective reagent for modification ofhistidine residues when the reaction is carried out at pH near neutral (4, 28). Its effect on the A vena proteases is illustrated in Figure 3. Strong inhibition of both is evident, indicating the presence of histidine at the active sites. Although tyrosine, tryptophan, and methionine are also attacked by rose bengal, the latter two react at appreciable rates Units pH 6.6 pH 4.2 Table II Inhibition of Avena leaf proteases by IAc and pOIB Hemoglobin Bovine serum albumin Gelatin Casein (insoluble) Azocoll 4.53 1.12 0.95 0.40 1.02 3.17 1.58 2.92 3.38 6.40 originally thought to be the more potent enzyme by virtue of its Preincubations at 30 C for indicated times prior to standard hemoglobin assay. Inhibition at Preincubation pH 4.2 pH 6.6 10 mM IAc, 30 min 10 mM IAc, 150 min 0.04 mM pCMB, 30 min 0.4 mM pCMB, 30 min 1.4 7.2 0 2.1 18.3 48.0 0 4.0 reactivity against hemoglobin, is actually less active than the neutral enzyme against other proteins (Table I). The most pronounced differences are apparent with gelatin, casein, and Azocoll, with which substrates the neutral activity is three to eight times as great. It is suggestive that the BSA and casein samples used were native proteins, whereas the hemoglobin was acid-denatured. Although the limited number of proteins tested makes generalizations only tentative, the apparent differences in susceptibility of native and denatured proteins raise interesting possibilities with regard to the in vivo significance of these proteases in senescence. This will be considered below. 2 Action of Inhibitors. Since previous experiments have indicated z that only the neutral protease requires a reduced sulfhydryl group z for its functioning (9), the effects of pCMB and lAc on enzyme A' activities were tested. Table II shows that the activity at pH 4.2 is only slightly affected by IAc, but the activity at pH 6.6, following a 3-hr preincubation, shows a 50% inhibition. Complete reaction of sulfhydryl groups with lAc often requires long incubation periods and conditions that involve protein denaturation (3), so the incompleteness of the inhibition is not surprising. Histidine residues are also susceptible to attack by lAc, but the relative rate of this reaction between pH 6 and 8 is extremely slow. .001 .01 .1 1.0 The lack of any significant inhibition by pCMB is an apparently PMSF CONCENTRATION (MxalO) contradictory result, but since this and other organic mercurials FIG. 2. Effect of PMSF on the activities of Avena proteases. Enzyme show very limited solubility in water, their approach to relatively was preincubated with the inhibitor in 5% ethanol for 30 min at 30 C. hydrophilic centers of proteins can be limited also. Furthermore, Controls contained 5% ethanol without PMSF. Hemoglobin assay, 50 C. recent experiments have shown that inhibition of the neutral protease by HgCl2 is partially reversible by the P-mercaptoethanol in the assay buffer, and this could be occurring in the case of pCMB as well. The reagent N-bromo-succinimide inhibits both enzymes, 1 mM preincubated with the enzyme for 30 min at 30 C pH 4. sufficing for 95% inhibition. However, the specificity of this re2 / 10ISO agent is not sufficiently narrow to allow for definite deductions. The same is true for the actions of mercury and silver salts, both of which were found to cause 60 to 100% inhibition at 10 mm. Both trypsin and chymotrypsin are powerfully inhibited by 20 PMSF, through its reactivity with the active serine hydroxyl; it z 6 also attacks the hydroxyl of acetylcholinesterase, though more slowly (5, 10). In addition, it retards the senescence of detached oat leaves (25). Figure 2 demonstrates effective inhibition by PMSF of both Avena proteases, with a slightly greater effect on the neutral enzyme. Whitaker and Perez-Villasenor (29) have clearly demonstrated inhibition of papain (a sulfhydryl protease) by PMSF, and this ability to attack free sulfhydryls probably explains the inhibition of the neutral protease. Since the neutral protease from Avena is stimulated by mercaptoethanol and inI I rt 4 0 hibited by IAc, while the acid protease is unaffected by these ".001 .1 1.0 .01 PMSF indicate that the obtained with the results compounds, ROSE BENGAL CONCENTRATION (%) neutral enzyme is sulfhydryl-dependent, and the acid enzyme is FIG. 3. Effect of rose bengal on the activities of A vena proteases. The hydroxyl-dependent. In contrast, the acid protease from germi- enzyme was irradiated under a spotlight in the presence of the dye for 5 nating sorghum seeds shows no sensitivity to either -SH or -OH min prior to assay. Controls contained the dye but were protected from Hemoglobin assay, 50 C. reagents (12). Downloaded from on June 18, 2017 -light. Published by www.plantphysiol.org - - Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 61, 1978 DRIVDAHL AND THIMANN 504 Table III Effects of metal ion chelators on activity of Avena leaf proteases The enzymes were incubated in chelator solutions for 30 min at 30 C prior to standard hemoglobin assay. Chelator Inhibition at: pH 6.6 pH 4.2 0 Na2EDTA, 1.0 mN 2.4 Na2EDTA, 10.0 mM a,a-Dipyridyl, 1.0 mM 0 a,a-Dipyridyl, 10.0 mM 0 41.1 45.0 15.3 52.4 only below pH 4, while tyrosine reacts only above pH 8 (18). The involvement of histidine may explain the slight inhibition of the acid protease by lAc (Table II). The evidence indicates that this enzyme contains no reactive sulfhydryl, and in the absence of this group lAc will attack histidine (5). However, the reactivity of the imidazole group toward lAc is low (14), and thus only a very small inhibition is produced. Table III shows the effects of two different metal ion chelators on protease activities. No inhibition of the acid protease was seen, and hence a metal ion of the type removable by these reagents is probably not involved in its function. Both EDTA and dipyridyl exerted a moderate inhibition of the neutral protease, but the concentrations required were higher than would be expected. In contrast, Vallee and Neurath (26) reported complete inhibition of bovine pancreatic carboxypeptidase by I mM EDTA or 5 mM dipyridyl. Since the effect of EDTA shown here is nearly maximal at I mm and is increased only slightly by a 10-fold increase in concentration, there may be a metal atom present which is not absolutely essential for activity. Alternatively, a small amount of contaminating metallo-protease may be present. A similar explanation for partial EDTA inhibition of a-amylase has been proposed by Jacobsen et aL (15). Evidence on Mechanism of Action. As seen in Figure 4, the nitrogenous products produced upon digestion of hemoglobin by the acid protease are of heterogeneous sizes; in contrast, digestion by the neutral enzyme produces essentially one size class, the elution volume of which closely approximates that of an amino acid standard. It is thus reasonable to designate the acidic enzyme as an endoprotease and the neutral one as an exoprotease. The acidic enzyme of mung bean cotyledons is also apparently an endoprotease (8). Another difference between the two proteases was seen in their attack on synthetic substrates. Table IV shows that the three arginine derivatives were more sensitive to the neutral than to the acid enzyme, while the tyrosine derivative was much more rapidly attacked by the acid enzyme. Such differences, as weli as that above, give some indication of how the rapid and extensive proteolysis and liberation of free amino nitrogen are achieved in leaf senescence. Neither enzyme was sensitive to the soybean trypsin inhibitor, even at a concentration of 500 mg/I. Evidence for Autllysis. It has been suggested that proteases present unique problems in protein purification, not only because of their tendency to aggregate with other proteins but more particularly because they may undergo autolysis in solution. That autolysis may occur in the enzyme preparations discussed here is clearly shown by Figure 5. In this experiment, a partially purified sample was chromatographed on Sephadex G- 100, and the active fractions were pooled and stored at 6 C for 3 days. They were then rechromatographed on the same column. Even though the final peak of material absorbing at 280 nm had been discarded after the first column run, a very similar peak appeared on the second run (upper figure). Since this peak consists of low mol wt peptides and amino acids, the results indicate proteolytic digestion of proteins in the enzyme preparation. Gel electrophoresis has shown that the proteases are almost the only components of the preparation at this stage (see Fig. 3 of ref. 9), and since a significant loss of activity is apparent, the renewed second peak most probably represents autolytically generated fragments. The occurrence of such autolysis supports the suggestion of Martin and Thimann (17) that the rapid decrease in protease activity toward the end of the senescence period in oat leaves resulted from self-degradation. DISCUSSION In any consideration of the regulation of proteolytic activity the problem of substrate susceptibility is crucial. Several proteins were degraded by the preparations from oat leaves, but at strikingly different rates. It has been suggested (19) that such differences in rate are due to the existence in some proteins of susceptible peptide bonds in exposed regions of the molecule which are characterized by a high degree of local flexibility, thus facilitating "fit" into the active center of the protease. Such susceptibility can be increased by the breakage of certain bonds which cause the polypeptide chain to unfold (20). It seems likely, therefore, that in vivo, the activity of one of the A vena proteases renders some of the leaf substrates more prone to attack by the other. Since the data of Figure 4 have shown that only the acidic enzyme is an endoprotease, its activity could produce fragments which are more readily reduced to amino acids by the exoprotease. Correspondingly, in the senescing leaf it is the acidic protease whose activity increases first, while that of the neutral protease develops more slowly (17). The A vena leaf proteases evinced rather broad specificities, in that ali proteins tested were degraded to some extent. Given the multiplicity of proteins to be degraded in a senescing leaf, this breadth of specificity would be an appropriate feature. In the previous report (9) we noted that mercaptoethanol stim- FRACTION NUIMER FIG. 4. Sephadex G-50 chromatography of acidic and neutral protease reaction products (hemoglobin assay). Column dimensions were 1.5 x 95 cm, eluting buffer was 0.02 M sodium phosphate (pH 7). Table IV Hydrolysis of synthetic substrates by Avena leaf proteases All substrates were used at 1.0 mM. Assay conditions as described in Methods. All data from linear proportionality ranges. Substrate HTydrolysis change pH 6.6 pH 4.2 &OD/hr Benzoyl-L-arginine ethyl ester Benzoyl-L-arginine p-nitroanilide Tosyl-L-arginine methyl ester Benzoyl-L-tyrosine ethyl ester Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1978 American Society of Plant Biologists. All rights reserved. 0.325 0.285 0.080 0.575 0.610 0.642 0.430 0.340 Plant Physiol. Vol. 61, 1978 PROTEASES OF SENESCING OAT LEAVES 1.2 3.0 0.6 2.0 ENZYME PROTEIN 1.0 0O4-A PROTEIN ol.2 Z ~ENZYME 'hi0 04 0.6 1. 505 from oat seedlings with a mol wt of about 60,000, but the preparations also contained a number of minor active species, having mol wt as low as 10,000; it was suggested that these were autolytically generated fragments. Similarly, at least some of the multiple forms of a protease from Rhizopus oligosporus are produced by autolysis (27). However, in the case of ficin (16) and a barley endopeptidase (7), autolysis was ruled out as an explanation for multiple forms; the existence of several distinct gene products was the preferred explanation. The continued generation of autolytic fragments in solution can create serious problems in enzyme purification and characterization. Yet, if such fragmentation occurs in vivo, with minimal loss of proteolytic activity, it would lend a useful diversity and stability to the process of protein degradation during senescence. LITERATURE CITED 20 40 60 30 FRACTION NUMBER FIG. 5. Test for autolysis of protease preparation by repeated G-100 chromatography of active fractions. Lower figure shows the chromatography of the extract at the G-5O stage. Right hand peak ("protein") was then discarded. Upper figure shows the rechromatography of the active fractions ("4enzyme"9) from the first column after 3 days of storage at 6 C at pH 6. Column dimensions were 1.5 x 90 cm, eluting buffer was 0.05 M sodium phosphate (pH 6), 3-mi fractions. Protease assay at pH 4.2 and SOC. ulated the activity of the neutral protease, whereas it had no effect on the acid one, except for a small inhibition at high concentrations. This stimulation, together with the inhibition of the neutral protease by both IAc and PMSF, strongly suggests that it is a sulfliydryl enzyme. Since the acidic enzyme is not affected by IAc, the inhibitory effect of PMSF on it is probably due to the acylation of an active site hydroxyl group. Attempts to implicate histidine residues in protease activity have frequently employed as specific labels the chloromethyl ketones of phenylalanine and lysine. In trypsin and chymotrypsin these compounds act by alkylating histidine residues at the active site (22). However, tosyl-lysylchloromethyl ketone alkylates cysteine in the active site of papain (2, 29), and hence the more specific reagent, rose bengal, was used in this study. Means and Feeney (18) considered photooxidation to be the best general procedure for selective histidine modification. The results shown here indicate a critical role for histidine in the functioning of both Avena proteases. No requirement for metal ions in catalysis by the acid protease is apparent, but the partial inhibition of neutral enzyme activity by EDTA and dipyridyl remains unexplained. It could be the result of the presence of a metal atom which accelerates hydrolysis but is not essential for it, or of the presence of a contaminating metallo-protease. However, no stimulation of activity was observed upon addition of various metal ions (Cu2+, Zn2+, Mg2+, Mn2e) to assay buffers at concentrations up to 10mt (results not shown). In view of this, it is unlikely that the previously reported inhibition of leaf senescence by chelators (24) is related to any effect on protease activity. The phenomenon of autolysis probably explains the previously reported shoulders in the pH curve for the acid proteases of oats (9) and of wheat (11). Pike and Briggs (21) purified a protease 1. ANDERSON JW, KS ROWAN 1965 Activity of peptidase in tobacco leaf tissue in relation to senescence. Biochem J 97: 741-746 2. ARNON R 1970 Papain. Methods Enzymol 19: 226-244 3. BATTELL ML, GG ZARKADAS, LB SMILLIE, NB MADSEN 1%8 The sulfsydryl groups of muscle phosphorylase. III. Identification of cysteinyl peptides related to function. J Biol Chem 243: 6202-6209 4. BELLIN JS, CA YANKUS 1968 Influence of dye binding on the sensitized photo-oxidation of amino acids. Arch Biochem Biophys 123: 18-28 5. BERNHARD S 1968 The Structure and Function of Enzymes. WA Benjamin, New York 6. BOYER PD 1954 Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. I Am Chem Soc 76: 4331-4337 7. BURGER WC 1973 Multiple forms of acidic endopeptidase from germinated barley. Plant Physiol 51: 1015-1021 8. CHRISPEELS MI, B BAUMGARTNER, N HARRIS 1976 Regulation of reserve protein metabolism in the cotyledons of mung bean seedlings. Proc Nat Acad Sci USA 73: 3168-3172 9. DRIVDAHL RH, KV THIMANN 1977 The protses of senescing oat leaves. I. Purification and general properties. Plant Physiol 59: 1059-1063 10. FAHRNEY DE, AM GOLD 1963 Sulfbydryl fluorides as inhibitors of esterases. I. Rates of reaction with acetylcholinesterase, a-chymotrypsin, and trypsin. I Am Chem Soc 95: 997-1000 11. FRITH GIT, DG BRUCE, MJ DALLING 1975 Distribution of acid protease activity in wheat seedlings. Plant Cell Physiol 16: 1085-1091 12. GARG GK, TK VIRUPAKSHA 1970 Acid protease from germinated sorghum. I. Purification and characterization of the enzyme. Eur I Biochem 17: 4-12 13. GOLDTHWAITE II 1967 Physiological investigations of leaf tissue senescence. PhD thesis. Univ California, Berkeley 14. HEINRIKSON RL, WH STEIN, AM CRESTFIELD, S MOORE 1965 The reactivities of the histidine residues at the active site of ribonuclease towards haloacids of different structures. J Biol Chem 240: 2921-2934 15. JACOBSEN IV, IG SCANDALIOS, IE VARNER 1970 Multiple forms of amylase induced by gibberellic acid in isolated barley aleurone layers. Plant Physiol 45: 367-371 16. JONES IK, AN GLAZER 1970 Comparative studies on four sulfhydryl endopeptidases ("ficins") of Ficus glabrata latex. I Biol Chem 245: 2765-2772 17. MARTIN C, KV THIMANN 1972 The role of protein synthesis in the senescence of leaves. I. The formation of protease. Plant Physiol 49: 64-71 18. MEANS GE, RE FEENEY 1971 Chemical Modification of Proteins. Holden Day, San Francisco 19. NASLIN L, A SPYRIDAKIs, F LATEYRIE 1973 A study of several bonds hypersensitive to proteases in a complex flavoheme-enzyme, yeast cytochrome bs. Eur I Biochem 34: 268-283 20. OTTESON M 1967 Induction of biological activity by limited proteolysis. Annu Rev Biochem 36: 55-76 21. PIKE CS, WR BRIGGS 1972 Partial purification and characterization of a phytochrome degrading neutral protease from etiolated oat shoots. Plant Physiol 49: 521-530 22. SHAw E 1970 Selective chemical modification of proteins. Physiol Rev 50: 244-296 23. SHIBAOKA H, KV THIMANN 1970 Antagonisms between kinetin and amino acids. Experiments on the mode of action of cytokinins. Plant Physiol 46: 212-220 24. TETLEY RM, KV THIMANN 1975 The metabolism of oat leaves during senescence. IV. The effects of a,a'-dipyridyl and other metal chelators on senescence. Plant Physiol 56: 140-142 25. THIMANN KV, H SHIBAOKA, C MARTIN 1970 On the nature of senescence in oat leaves. In DI Caff, ed, Plant Growth Substances 1970. Springer-Verlag, Berlin, pp 564-573 26. VALLEE BL, H NEURATH 1955 Carboxypeptidase, a zinc metalloenzyme. I Biol Chem 217: 253-261 27. WANG HL, CW HESSELTINE 1970 Multiple forms of Rhizopus oligosporus protease. Arch Biochem Biophys 140: 459-463 28. WESTHEAD EW 1965 Photo-oxidation with rose bengal of a critical histidine residue in yeast enolase. Biochemistry 4: 2139-2144 29. WHITAKER JR, i PEREZ-VILLASENOR 1968 Chemical modification of papain. I. Reaction with the chloromethyl ketones of phenylalanine and lysine and with phenylmethylsulfonyl fluoride. Arch Biochem Biophys 124: 70-78 Downloaded from on June 18, 2017 - Published by www.plantphysiol.org Copyright © 1978 American Society of Plant Biologists. All rights reserved.