Plant Physiol. (1981) 67, 110-1140032-0889/81/67/01 10/05/$00.50/0

Hemoglobin-digesting Acid Proteinases in Soybean LeavesCHARACTERISTICS AND CHANGES DURING LEAF MATURATION AND SENESCENCE1

Received for publication May 5, 1980 and in revised form July 24, 1980

LAVERNE E. RAGSTER2 AND MAARTEN J. CHRISPEELS3Department of Biology, C-016, University of California, San Diego, La Jolla, California 92093

ABSTRACT

Three proteinases which digest hemoglobin rapidly at acid pH (3.5 to4.5) were identified in crude extracts of soybean (Meff.) leaves andseparated by chromatography on DEAE-cellulose. All three enzymes wereendopeptidases as judged by the ratio of a-amino-nitrogen plus peptidenitrogen over a-amino-nitrogen in the trichloroacetic acid-soluble portionof hemoglobin digests. Proteinase I did not bind to diethylaminoethylcellulose and was not inhibited by any of the proteinase inhibitors tested.Proteinase II was partially inhibited by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and p-chloromercuribenzoate. The inhibition by phenyl-methylsulfonyl fluoride can probably be accounted for by the presence ofcontaminating carboxypeptidase. Proteinase III was the most anionic ofthe three and required the presence of sulfhydryl reagents to prevent theirreversible loss of activity. All the proteinase preparations digested soy-bean ribulose bisphosphate carboxylase as shown by the disappearance ofthe large subunit of that protein, when partially digested preparations weresubjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gels.These experiments confirmed that the three proteinases were endopepti-dases. All three proteinases were present throughout leaf development;proteinase I predominated in expanding leaves, whereas proteinase IIIbecame the predominant enzyme as the leaves matured. Senescence (yel-lowing) was associated with a decline in the activities of all three protein-ases.

A number of studies have been made in recent years to elucidatethe control of leaf protein catabolism associated with leaf senes-cence and seed formation (4, 7, 10, 22). Most efforts have beendirected towards finding out whether there are "senescence-spe-cific" proteinases, i.e. proteinases which increase in activity at thetime of leaf senescence. The discovery of such proteinases wouldprovide a starting point for further investigation of leaf proteincatabolism during senescence. Some investigators have providedevidence that leaf proteinases increase in activity at the time ofleaf senescence (4, 7, 22), whereas others could find no suchtemporal correlation (1, 14, 17). These discrepancies may wellhave resulted from the examination of different species, as well asof different proteinases, by the various researchers. It is apparentthat proteinases have marked substrate specificity and proteinasesactive on one substrate (e.g. Azocoll) may be largely inactive on

'This work was supported by grants from the Herman Frasch Foun-dation, the National Science Foundation (Developmental Biology), andthe United States Department ofAgriculture (Competitive Research GrantOffice).

2Present address: Division of Mathematics and Science, College of theVirgin Islands, St. Thomas, VI 00801.

3 To whom reprint requests should be addressed.

another one (e.g. hemoglobin).The leaves of many plants contain proteinases which are max-

imally active between pH 3.5 and 5.5 and are capable of digestinganimal proteins, such as casein, Azocoll, or hemoglobin (6-8), aswell as plant proteins, such as RuBPCase4 (11, 12, 21, 22). Indirectevidence indicates that such acid proteinases may play an impor-tant role in protein breakdown during leaf sensecence (4, 7, 21,22). Peoples et al. (12) showed that there is a correlation betweenthe activity of such acid proteinases, which digest hemoglobin aswell as RuBPCase and the rate of loss of protein-nitrogen fromthe same vegetative organs of wheat.We have examined some proteinases with basic pH optima

present in soybean leaves (14) and here describe three acid pro-teinases which digest both hemoglobin and RuBPCase. One ofthem increases markedly in activity when the leaves reach matu-rity, but none fits into the category of "senescence-specific" pro-teases.

MATERIALS AND METHODS

Plant Material. Soybeans [Glycine max (L.) Merr. cv. Steele]were grown in pots in the greenhouse without supplementallighting in La Jolla, CA. Prior to planting, the seeds were treatedwith Nitragin Rhizobium inoculum. Leaf samples were collectedas needed from the third, fourth, or fifth trifoliate node, kept onice, and transported to the laboratory. Leaf samples for long-termdevelopmental studies were kept at -20 C after harvest. For thebiochemical characterization experiments, leaves defined as youngare those which have just expanded, those defined as mature areat least 2 weeks past full expansion, and those defined as senescentshow the first signs of yellowing.

Extraction. Leaves were cut into small pieces and homogenizedin a Polytron homogenizer (Kinematica, Luzern, Switzerland), for90 to 120 s at 3 C in 4 volumes extraction medium. The extractionmedium consisted of 50 mm K-phosphate (pH 7.5) with 1%insoluble PVP (Sigma) and 20 mm sodium metabisulfite. Thehomogenate was filtered through four layers of cheesecloth andcentrifuged for 10 min at 12,000g. The supernatant was dialyzedagainst 25 mm K-phosphate (pH 7.5) for 3 to 5 h. The dialyzedsupernatant was used for protein and enzyme assays. Protein andChl levels were determined as described (14).

Proteinase Assay. Aliquots of dialyzed extracts or DEAE col-umn fractions (100 to 200 ,ul) and 250 ,l hemoglobin (6 mg/mlSigma bovine type I) dissolved in 200 mm Na-acetate (pH 4) wereincubated for 90 to 120 min at 45 C in a shaking water bath.Reactions were terminated with trichloroacetic acid at a finalconcentration of 5%. After 30 min at 3 C, the trichloroacetic acid-soluble a-amino-N was measured colorimetrically with ninhydrin

'Abbreviations: RuBPCase, ribulose bisphosphate carboxylase; LS,large subunit of RuBPCase; PMSF, phenylmethylsulfonyl fluoride; NEM,N-ethylmaleimide.

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ACID PROTEINASES IN SOYBEAN LEAVES

(16), using less than 250 ,ul of the trichloroacetic acid supernatant.Inhibitor Studies. Dialyzed extracts were pretreated with inhib-

itors for 12 h at 3 C. Controls were aliquots of extracts pretreatedwith an equivalent volume of solvent used for the inhibitor.Column Chromatography. Crude extract was adsorbed on a

DEAE-cellulose (Whatman, DE32) column (20 x 1.5 cm) equili-brated with 25 mm K-phosphate buffer (pH 7.5) containing 2 mm2-mercaptoethanol. The column was washed with 50 ml equilibra-tion buffer, and the bound proteins then were eluted with a 0 to0.7 M KCI gradient in equilibration buffer. The fractionation wascarried out at 5 C.

SDS-Polyacrylamide Gel Electrophoresis. Polypeptides wereseparated by SDS-polyacrylamide gel electrophoresis by themethod of Laemnli (9) slightly modified (2). Samples which hadbeen precipitated with trichloroacetic acid were first extractedwith 80% acetone to remove excess acid. Pellets extracted in thismanner, or mixtures of partially digested proteins and peptides(not treated with trichloroacetic acid), were dissolved in denatur-ation buffer [2% SDS, 0.3% 2-mercaptoethanol, and 50 mm Tris-HCI (Sigma, pH 8)] and boiled for 4 min. The gels were stainedwith Coomassie brilliant blue and destained. To determine therelative amount of the large subunit of RuBPCase, the destainedgels were scanned with a Joyce-Loebl microdensitometer, and thearea under the peak was measured by weighing the paper. Allcomparisons between samples (e.g. effect of pH or time of incu-bation) were done with samples from one slab gel.

Ratio of a-amino-N plus Peptide Bond-N to a-amino-N inTrichloroacetic Acid Soluble Hydrolysis Products. Aliquots of thepartially purified enzymes separated by chromatography onDEAE-cellulose were incubated with hemoglobin as described,and the undigested protein was precipitated with trichloroaceticacid. After standing overnight at 0 C, the supernatants werecollected by centrifugation and extracted three times with 4 vol-umes diethyl ether to remove the trichloroacetic acid. Half of eachsample was used for a determination of free a-amino-N (16). Theother half was mixed with an equal volume of 12 N HCI, sealed ina vial, and heated to 105 C for 18 h. The HCI was removed witha rotary evaporator and the residue was dissolved in H20. Analiquot was used to determine total a-amino-N (16). The ratio ofa-amino-N after acid hydrolysis to a-amino-N before acid hy-drolysis is a measure of the contribution of exo- and endopepti-dases to the digestion of the proteins.

Digestion of LS of RuBPCase. A measure of endopeptidicproteolysis was obtained by measuring the relative intensity ofstaining of the LS of RuBPCase on polyacrylamide gels. Purifiedcarboxylase (200 ,ug) was incubated with aliquots of partiallypurified hemoglobinases I, II, and III for 1 or 2 h at 37 C. The pHwas adjusted with 100 ,ld 0.2 M buffer (citrate-phosphate for pH3.5 to 7.0; Tris for pH 7.0 to 9.5). The partially digested sampleswere precipitated with trichloroacetic acid (final concentration,7.5%), and the precipitated polypeptides were separated by SDS-polyacrylamide gel electrophoresis as described.To obtain purified RuBPCase, mature soybean leaves were

homogenized in 25 mm Hepes buffer (pH 7.5) containing 1 mmEDTA and 4 mM DTT. The homogenate was centrifuged for 20min at 27,000g and a l-ml aliquot was loaded on a linear sucrosegradient (5-25% w/w in the same buffer). The gradients werecentrifuged for 17 h at 150,000g and collected. The large peaknear the bottom of the gradient was collected and consistedentirely of RuBPCase; it was judged to be free of contaminatingproteins by SDS-polyacrylamide gel electrophoresis.

RESULTS

Separation and Characterizations of Hemoglobin-digesting En-zymes. Crude extracts ofsoybean leaves clarified by centrifugationcontain proteinase(s) which digest hemoglobin over a wide pHrange (Fig. 1). The pH curve is biphasic with a maximum at pH

8-

6-

4n

.E42-

3 4 5 6 7 8 9 10 11pH

FIG. 1. Effect of pH on hemoglobin-digesting activity in crude extractsof soybean leaves. Crude extracts were dialyzed against 50 mm buffers(Na-acetate, pH 5.0; K-phosphate, pH 7.5; Tris, pH 9.0) and the pH of 10-ml aliquots was further adjusted to the values indicated. Aliquots thenwere incubated with hemoglobin and the a-amino-N in the trichloroaceticacid supernatant was estimated with ninhydrin. The activity is expressedas ,Lmol leucine/ml *h.

94.

!T

I

2

-

L.

Fraction Number

FIG. 2. Fractionation of hemoglobin-digesting proteinases on DEAE-cellulose. Clarified leaf extract was loaded on a DEAE-cellulose column(20 x 1.5 cm) equilibrated with 25 mM K-phosphate (pH 7.5) containing2 mm 2-mercaptoethanol. The column was washed with 100 ml equilibra-tion buffer and then eluted with a gradient of 0 to 0.7 M KCI. Fractionswere collected and assayed for protein and hemoglobin-digesting activityat pH 4.5.

5 and a second one at pH 7.5. A similar pH curve was obtainedwhether extracts of mature or senescent leaves were used. Thehigher activity in the acid pH range (4.0 to 5.0) and the observationthat the incubation of leaf extracts in this pH range causes therapid hydrolysis of the LS of RuBPCase (15) led us to examinethe acid hemoglobinases further. All subsequent assays for he-moglobinase activity were carried out at pH 4.0 or 4.5.The proteins present in crude extracts clarified by centrifugation

were fractionated by chromatography on DEAE-cellulose andthree peaks of hemoglobin-digesting activity were obtained (Fig.2). Proteinase I eluted with the unabsorbed proteins, proteinase IIeluted in the same region of the gradient as carboxypeptidase andaminopeptidase (data not shown here, but see Fig. 4 of ref. 14),and proteinase III only eluted off the column if the final saltconcentration was at least 0.7 M.

Since many proteinases require sulfhydryl reagents for maximalactivity, parallel columns were run with and without 2 mm mer-

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RAGSTER AND CHRISPEELS

captoethanol in the elution buffer. Omission of the mercaptoeth-anol resulted in very low (less than 20%) activity of proteinase III,but there was no change in proteinases I and II (data not shown).Subsequent addition of mercaptoethanol to the column fractionsprior to assaying did not restore the activity of proteinase III,indicating that proteinase activity has been irreversibly lost. Over-night dialysis at 4 C of the fractions containing proteinase IIIagainst a buffer containing less than 2 mm mercaptoethanol alsoresulted in an irreversible loss of activity.The pH optima for the three proteinases were determined

separately with hemoglobin as a standard (Fig. 3). The data showthat all three enzymes are most active at pH 4.0, and subsequentassays were done at this pH. Proteinase III has considerableactivity at pH 5.5 to 6.5 or is contaminated with another proteinaseactive in that range.

Since proteinase II was known to be contaminated with carbox-ypeptidase and aminopeptidase, we assessed the contributions ofexo- and endopeptidases to the measured activity for all threeproteinases. This was done by determining the ratio of a-amino-N plus peptide-N to a-amino-N in the trichloroacetic acid super-natant of the partially digested hemoglobin. We obtained ratios of9.3, 3.7, and 6.6 for proteinases I, II, -and III, respectively. Thesedata indicate that the activities in peaks I and III are due largelyto endopeptidase, whereas some of the activity in peak II may bedue to exopeptidase, probably carboxypeptidase. Indeed, carbox-ypeptidase has a pH optimum of 5.0 and is quite active at pH 4.0,whereas aminopeptidase (assayed with leucyl p-nitroanilide assubstrate) showed essentially no activity at pH 4.0 (data notshown).The sensitivity of the three proteinases to inhibitors of protein-

ase activity differed (Table I). EDTA (3 mM) did not affect any ofthe enzymes. PMSF (2 mM) affected only proteinase II (58 %inhibition) indicating again that proteinase II activity may be duein part to carboxypeptidase. Indeed, plant carboxypeptidase hasbeen shown to be inhibited by proteinase inhibitors, such as PMSFand diisopropyl phosphofluoridate, which affect proteinases witha serine residue at or near the active site. The sulfhydryl reagentsp-chloromercuribenzoate and NEM did not inhibit proteinase Iand inhibited proteinase III more strongly than proteinase II.

2.5

m2.0H\.1.

J.-

1.5-

3 4 5 6 7 8 9 10pH

FIG. 3. Activity of proteinases I, II, and III at different pH values. Leafextracts were fractionated as in Figure 2, and the three peaks of proteinaseactivity were combined, dialyzed, and concentrated with Aquacide. Ali-quots (250 ,tl) of the concentrated proteinase were incubated with hemo-globin (250,l) at the desired pH (200 mm citrate-phosphate for pH 3.0 to7.5, and 200 mM Tris for pH 7.5 to 9.5). a-Amino-N in the trichloroaceticacid supernatants of the digests was estimated with ninhydrin and ex-pressed as ,umol leucine/mg protein. h.

Table I. Inhibition ofActivities of Hemoglobin-digesting Proteinases I, II,and III

DEAE column fractions corresponding to hemoglobin-digesting activi-ties I, II, and III were combined and dialyzed for 2 h against 25 mm K-phosphate (pH 7.5). Aliquots of extracts were preincubated with inhibitorsfor 12 h at 3 C. Measurements of residual hemoglobinase activity weremade as described.

Inhibition of Hemoglobin-digesting

Treatment Concentra- Proteinasetion

I- II IIImM %

EDTA 3.0 0 0 0PMSF 2.0 0 58 0p-Chloromercuri- 0.1 11 31 62

benzoateNEM 0.5 14 73NEM 2.5 4 62 75

Proteinase III, which lost most activity upon prolonged dialysis inthe absence of 2-mercaptoethanol was inhibited 73% by 0.5 mmNEM.Temporal Changes during Leaf Development. To establish the

developmental patterns of these three enzymes during leaf ontog-eny, we measured temporal changes in total Chl, total protein,and hemoglobin-digesting activity in crude extracts of the thirdtrifoliate leaf of greenhouse-grown plants (Fig. 4A). The first timepoint (50 days after planting) represents half-expanded leaves andthe last time point (110 days after planting) represents yellowedleaves. Chl and protein/leaf increased 2- and 4-fold, respectively,to maxima reached about 75 days after planting and declinedsubsequently. Total hemoglobin-digesting activity/leaf increased3-fold as the leaves reached maturity and then declined. Activityremained relatively high during the time the leaves senesced andlost protein, resulting in a high specific activity during the laststages of senescence (Fig. 4B). The experiment was repeated withfield-grown plants (cv. Williams, grown at the Agronomy Farm ofthe University of Illinois, Urbana, IL) and essentially the sameresults were obtained. For that experiment, we used the ninthtrifoliate leaf and observed the same pattern of change, althoughthe absolute values for Chl, protein, and hemoglobin-digestingactivity were 2 to 3 times higher/leaf.Temporal changes in proteinases I, II, and III were determined

by subjecting dialyzed leaf extracts to fractionation with DEAE-cellulose. Three developmental stages were chosen for both thegreenhouse- and field-grown materials: half-expanded, mature,and yellowing. The same results were obtained with greenhouse-and field-grown materials and, again, only the results for green-house-grown material are reported (Fig. 5). The results clearlyshow that proteinase I is the predominant enzyme in half-ex-panded leaves (Fig. SA) and that these leaves have very littleproteinase III. The accumulation of proteinase III in the leaves isassociated with maturity (Fig. 5B); all proteinases decline inactivity during senescence (Fig. 5C), but the proteinase pattern ofsenescent leaves resembles that of mature leaves.To find out if the three proteinases could degrade native

RuBPCase, we purified RuBPCase from soybean leaves, incu-bated it in the presence of the partially purified proteinases, andseparated the products by SDS-polyacrylamide gel electrophore-sis. The decrease in staining intensity of the large subunit ofRuBPCase was taken as measure of RuBPCase digestion (15, 18).The rate of breakdown was constant until 40% of the LS ofRuBPCase had been lost. Thereafter, the rate of breakdownslowed and leveled off (data not shown). Incubation of RuBPCasewith each one of the three proteinases at different pH valuesindicated that all three enzymes degraded RuBPCase most rapidly

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Plant Physiol. Vol. 67, 1981

1 9-

0a#.7

U'

a.0LUc 3

. . .

25

20F

1._.

c

0

EG 5

ACID PROTEINASES IN SOYBEAN LEAVES

A -30

4-

6I

2-J

-20

0~~~~~~~~~~~.G@ I

.210 %J~~~

u.i St &

_ ~~I

.341.Sx4

4

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20Fraction Number

FIG. 5. Fractionation of hemoglobin-digesting proteinases present inextracts of young (A), mature (B), and senescent (C) soybean leaves.Conditions were as in Figure 2. All leaves were third trifoliate leavesharvested 48 days after planting (young, half-expanded), 74 days afterplanting (mature) and 88 days after planting (senescent, yellowing).

1

I="..

a.

0._

LA

I .50 70 90 120

DAYS AFTER PLANTINGFIG. 4. Temporal changes in; A, protein ( *) and Chl (A A)

content; and B, hemoglobin-digesting activity per leaf (Q-O) andspecific activity ( -) in crude extracts of the third trifoliate leaf ofgreenhouse-grown soybeans. Hemoglobin digesting activity is expressed as

,umol leucine/ml * h and specific activity is expressed as ,umol/mg proteinh.

at pH 3.5 to 4.5 (Fig. 6). Enzyme I or II had very little activity atpH 5.0 to 8.0, whereas proteinase III had considerable activity inthis pH range. Whether this was due to the presence of anotherenzyme contaminating the acid proteinase is not clear. There wasno breakdown of the LS of RuBPCase at any of the pH valuestried in the absence of added proteinase. Whether the breakdownobtained at pH 9.0 with enzyme I or II represents activity of adifferent enzyme at this basic pH remains to be determined.

DISCUSSION

Soybean leaves contain numerous proteinases. In a previousreport (14), we described two Azocoll-digesting enzymes whichare most active in the basic pH range (pH 9.0). Here, we report onthe presence of three acid proteinases which digest both hemoglo-bin and RuBPCase most rapidly around pH 3.5 to 4.5. Such acidproteinases have also been found in the leaves of wheat (8, 12,

pH

FIG. 6. Effect of pH on the digestion of RuBPCase by proteinase I, II,

or III. Proteinases present in extract of mature leaves were fractionated onDEAE-cellulose as in Figure 2; after dialysis and concentration withAquacide, aliquots (125 I1) of proteinase, RuBPCase (200 ,jg of protein in25 ,ul), and buffer (100ll) were incubated for 60 min at 37 C. Buffers were0.2 M citrate-phosphate (pH 3.5 to 7.5) or 0.2 M Tris (pH 7.5 to 9.5).Polypeptides were separated by SDS-polyacrylamide gel electrophoresisand the gel was stained with Coomassie blue. The amount of proteinremaining in the LS of RuBPCase was determined by scanning the gelwith a densitometer and the value compared with the amount initiallypresent. I, II, and III refer to the three proteinases in order of elution fromthe DEAE-cellulose column.

113

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RAGSTER AND CHRISPEELS

21), oats (5, 10), and corn (7).Chromatography on DEAE-cellulose partially purified the three

proteinases. Proteinase I was not bound to the column and elutedwith the Azocoll-digesting proteinases described earlier (14). Itcan be distinguished from the latter by its pH optimum andsensitivity to EDTA. The major Azocoll-digesting proteinase istotally inhibited by 3 mm EDTA, whereas the hemoglobin-digest-ing proteinase I is unaffected by it or any other inhibitor tried. Itsendopeptidase character is verified by the high ratio of a-amino-N plus peptide-N to a-amino-N in the trichloroacetic acid solublehydrolysis products and by the breakdown ofthe LS ofRuBPCase.

Proteinase II eluted from the DEAE in the same region as thetwo exopeptidases leucine aminopeptidase and carboxypeptidase.Partial inhibition of the activity by PMSF and a low ratio of a-amino-N + peptide-N to a-amino-N in the trichloroacetic acidsoluble hydrolysis products indicated that the observed activity atpH 4.0 was due in part to the presence of carboxypeptidase.Carboxypeptidase from leaves of different plants (13, 20) andfrom soybean leaves (L. Ragster, unpublished) is inhibited byPMSF, whereas aminopeptidase had no activity at pH 4.0 (L.Ragster, unpublished).

Proteinase III was the most anionic enzyme, requiring 0.7 MKCI to remove it from the DEAE-cellulose. The activity wasirreversibly lost if sulfhydryl reagents, such as 2-mercaptoethanol,were omitted from the elution buffer and there was considerableinhibition of activity by 0.1 mm p-chloromercuribenzoate and 0.5mM NEM. These observations indicate that proteinase III isprobably a sulfhydryl enzyme. The endopeptidic character of thisenzyme was also confirmed by the high ratio of a-amino-N +peptide-N to a-amino-N in the trichloroacetic acid soluble hy-drolysis products and by the cleavage of the LS of RuBPCase.Temporal changes in the activity of the three proteinases during

leaf ontogeny indicate that all three enzymes were presentthroughout but that a dramatic increase in proteinase III activitywas associated with leaf maturity. Senescence (loss of protein andyellowing) was generally accompanied by a loss of total hemoglo-bin-digesting activity and a loss of the three proteinases. Althougha number of studies have shown that proteinase activity increaseswhen leaves senesce (4, 7, 22), an equal number of studies showsenescence is not to be accompanied by a rise in proteinase activity(1, 14, 17). It is not yet clear whether the increase in proteinaseactivity which accompanies senescence in some species is a pre-requisite for leaf protein breakdown. A recent study (19) withexcised oat leaves shows that the rise in proteinase activity, whichnormally accompanies excision and yellowing when the leaves arefloated on H20, does not occur when the leaves are placed upright.However, protein breakdown occurred to the same extent in bothsets of leaves. Such studies indicate that the level of proteinaseactivity is not the controlling factor in leaf protein breakdownduring senescence. In general, protein breakdown may not dependon the synthesis of senescence specific proteases, even thoughsenescence may often by accompanied by an increase in proteinaseactivity.Another manner in which the degradation of leaf protein may

be regulated is by changes in compartmentation, of which little isknown. Acid proteinases have been found in the vacuole (3) butwe do not know whether the hemoglobin-digesting proteinasesdescribed here are vacuolar enzymes. Experiments with isolated

chloroplasts show that there is no hemoglobin-digesting activityassociated with intact chloroplasts (15). If the hemoglobin-digest-ing proteins are located in the vacuole and if they are involved inthe in situ breakdown of intrachloroplastic proteins such asRuBPCase, it will be necessary to demonstrate how the vacuolarenzymes come in contact with their substrates in the chloroplasts.

Acknowledgments-We thank Ms. Bradford for technical assistance. It is a pleasureto thank R. H. Hageman and J. Harper for their help in growing soybean plants onthe Agronomy Farm at the University of Illinois, collecting the leaves, and shippingthem to San Diego.

LITERATURE CITED

1. ANDERSON JW, KS ROWAN 1966 The effect of 6-furfurylaminopurine on senes-cence in tobacco-leaf tissue after harvest. Biochem J 98: 401-404

2. BOLLINI R, MJ CHRISPEELS 1978 Characterization and subcellular localization ofvicilin and phytohemagglutinin, the two major reserve proteins of Phaseolusvulgaris L. Planta 142: 291-298

3. BUTCHER H, GJ WAGNER, HW SIEGELMAN 1977 Localization of acid hydrolasesin protoplasts. Examination of the proposed lysosomal function of the maturevacuole. Plant Physiol 59: 1098-1103

4. DALLING JM, GB BOLAND, JH WILSON 1976 Relation between acid proteinaseactivity and redistribution of nitrogen during grain development in wheat. AustJ Plant Physiol 3: 721-730

5. DRIVDAHL RH, KV THIMANN 1977 Proteases of senescing oat leaves I. Purifi-cation and general properties. Plant Physiol 59: 1059-1063

6. DRIVDAHL RH, KV THIMANN 1978 Proteases ofsenescing oat leaves. II. Reactionto substrates and inhibitors. Plant Physiol 61: 501-505

7. FELLER UK, TT SOONG, RH HAGEMAN 1977 Leaf proteolytic activities andsenescence during grain development of field grown corn. Plant Physiol 59:290-297

8. FRITH GJT, KHJ GORDON, MJ DALLING 1978 Proteolytic enzymes in greenwheat leaves I. Isolation on DEAE-cellulose of several proteinases with acidpH optima. Plant Cell Physiol 19: 491-500

9. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

10. MARTIN C, KV THIMANN 1972 The role of protein synthesis in the senescence ofleaves I. The formation of protease. Plant Physiol 49: 64-71

11. PEOPLES MB, MJ DALLING 1978 Degradation of ribulose-1,5-bisphosphate car-boxylase by proteolytic enzymes from crude extracts of wheat leaves. Planta138: 153-160

12. PEOPLES MB, GJT FRITH, MJ DALLING 1979 Proteolytic enzymes in green wheatleaves. IV. Degradation of ribulose-1,5-bisphosphate carboxylase by acid pro-teinases isolated on DEAE-cellulose. Plant Cell Physiol 20: 253-258

13. PRESTON, KR JE KRUGER 1976 Purification and properties of two proteolyticenzymes with carboxypeptidase activity in germinated wheat. Plant Physiol 58:516-520

14. RAGSTER L, MJ CHRISPEELS 1979 Azocoll-digesting proteinases in soybean leaves:characteristics and changes during leaf maturation and senescence. PlantPhysiol 64: 857-862

15. RAGSTER L, MJ CHRISPEELS 1981 Autodigestion in crude extracts of soybeanleaves and isolated chloroplasts as a measure of proteolytic activity. PlantPhysiol 67: 104-109

16. SPIES, JR 1957 Colorimetric procedures for amino acids. Methods Enzymol 3:468-471

17. STOREY R, L BEEVERS 1977 Proteolytic activity in relationship to senescence andcotyledonary development in Pisum sativum L. Planta 137: 37-44

18. VAN HUYSTEE RB 1973 A study on the degradation of arachin by proteolyticenzymes. Can J Bot 51: 2217-2222

19. VAN LOON LC, AJ HAVERKORT, GJ LOKHORST 1978 Changes in protease activityduring leaf growth and senescence. FESPP Abstr 280-C: 544-545

20. WELLS JRE 1965 Purification and properties of a proteolytic enzyme from frenchbeans. Biochem J 97: 228-235

21. WITTENBACH VA 1978 Breakdown of ribulose bisphosphate carboxylase andchange in proteolytic activity during dark-induced senescence of wheat seed-lings. Plant Physiol 62: 604-608

22. WITTENBACH VA, RC ACKERSON, RT GIAQUINTA, RR HERBERT 1980 Changesin photosynthesis, ribulose bisphosphate carboxylase proteolytic activity, andultrastructure of soybean leaves during senescence. Crop Sci 20: 225-231.

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