proteins and plant cell walls. proline to hydroxyproline ... · determination h3bo3 1.5 glycine 5...

BAUR AND WORKMAN-CELL PERMEABILITY AND RESPIRATION

diminished and when ripe both respiration and ionleakage were inhibited.

Literature Cited

1. BIALE, J. B. 1960. Respiration of fruits. p 548.Encyclopedia of Plant Physiology, W. Rhuland, ed.vol XII, pt 2. Springer-Verlag, Berlin.

2. BLACKMAN, F. F. AND P. PARIJA. 1928. Analyticstudies in plant respiration. I. The respiration ofa population of senescent ripening apples. Proc.Roy. Soc. (London), B. 103: 412-45.

3. LEONARD, E. R. AND C. W. WARDLAW. 1941. Stud-

ies in tropical fruit. XII. The respiration ofbanana during storage at 53F and ripening at con-trolled temperatures. Ann. Botany (NS) 5: 379-423.

4. SACHER, J. A. 1962. Relations between changes inmembrane permeability and the climacteric in ba-nana and avocado. Nature 195: 577-78.

5. THOMAS, M., S. L. RANSON, AND J. A. RICHARDSON.1958. Plant Physiology. 4th ed. J. and A.Churchill, Ltd., London. p 134.

6. YOUNG, R. E., R. L. BIELESKI, AND J. B. BIALE. 1961.Phosphate esterificat-ion in tissue slices of Fuerteavocado fruit. Plant Physiol. 36: xxx.

Proteins and Plant Cell Walls. Proline to Hydroxyproline inTobacco Suspension Cultures" 2,3

Alfred C. OlsonWestern Regional Research Laboratory,4 Albany 10, California

Hydroxyproline occurs in higher plants in pep-tides and proteins, which are found in the plant cellwalls (6, 9, 18, 19). Considerably more hydroxypro-line is found in the protein of rapidly proliferatingtissue than in the protein of slowly proliferating tissue(2, 7, 19). Hydroxyproline-rich plant protein andcollagen are similar in the large amounts of prolineand hydroxyproline they contain. The function of hy-droxyproline-rich plant protein may thus be structural,similar to that of collagen in animals, or perhapsstructural and regulatory as suggested by Lamport(8). In this connection Lamport has proposed thatsuch hydroxyproline-rich plant proteins be called"extensin" (8).

The conversion of proline to hydroxyproline isvery similar in animal and plant systems. In bothsystems free proline, but not free hydroxyproline, isa precursor of protein bound hydroxyproline (19).In general hydroxyproline-containing proteins aremetabolically inert (2, 11). Hydroxyproline hy-droxyl 02 of both animal and plant protein is derivedby the fixation of atmospheric 02 (3, 4, 8, 17).

Cultured plant cells are ideal for study of the

'Received Nov. 12, 1963.2 Work conducted under a cooperative agreement with

the California Institute of Technology, at the Division ofBiology, Pasadena, Calif.

3 Reported in part at the Western Section meeting ofthe American Society of Plant Physiologists, June 17 to21, 1963, Stanford University.

4A laboratory of the Western Utilization Researchand Development Division, Agricultural Research Service,United States Department of Agriculture.

location, function, and biochemistry of hydroxypro-line-containing plant protein because of the largeamounts of the protein present. This paper presentsresults of an investigation of the hydroxyproline-con-taining protein of the cell walls of suspension culturesof tobacco cells grown on a completely defined medi-um. As reported by others, these cells rapidly in-corporate proline-C'4 into all protein fractions asproline-C14 or hydroxyproline-C14. Data are pre-sented which suggest that in this system proline maybe hydroxylated after incorporation into a protein orlarge peptide.

Materials and MethodsTobacco Suspension Cultures. Suspension cul-

tures of tobacco cells were grown in a completelydefined liquid medium developed by Mr. Philip Filnerand described in table I (private communication).The cells were provided by Mr. Filner who originallyisolated them from tobacco stem callus (Nicotianatabacum var. Xanthi).

Cultures were grown from 5 to 10 days at 27 to280 in liter flasks on a platform-type reciprocal shakeroperating at 100 cpm with a horizontal excursion of4.5 cm.

Collection of the Cells and Their Fractionation.Cells were collected by filtering suspensions throughMiracloth5, a porous paper, and squeezing them free

5 Reference to a company name does not imply ap-proval or recommendation of the product by the Depart-ment of Agriculture to the exclusion of others that mayalso be suitable.

543

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Table 1. Comiipositioni of Cultuire Mediumt collected was 50 % protein by nitrogen analysis.Cell-wall residualprotein is that which remains asso-

Component mg/liter Component mg/liter ciated with the cellwvalls after sodium hydroxidleextractions.

Ca (NO) 24H O 200 i.-Arginine Sxrctos

KNOI)23 2 80 L-Lysine HCl 13 Incorporation of Proline-C'4 into the Cells. TheNaH2PO4H23 0 16.5 -Serine 10 turnover of proline and hydroxyproline in protoplasmlNa2SO4 200 L-Threoninle 8 and cell-wall protein fractions nleasured byinlcu-MgSO4 7HO 360 L-Cysteine 7 bating cells in amle(liumii containingproline-C'4 for

KCl 65 L-Methioniine 7 half an hour (the pulse) after which the specificFe citrate 2 L-Phenylalanine 32 activity of the exogenous labeled proline was loweredMnSO4 4H90 4.5 L-Tryptophan 4 by a. factor of 1000 ornmore by floo(ling themeliunmZnSO4 7H2O 1.5 L-Histidine 11 with unlabeled proline (the chase). DeterminationH3BO3 1.5 Glycine 5 of the specific activity of the proline and hydroxypro-KI 0.75 L-Alaine 8 line in the protein fractions of the cells as a functionNiacini 0.5 L-Valine 8Pyridoxiine 0.1 L-Leucinie 5 ot tinme then yielded somie informatioon about the rate

Thiamin 0.1 L-Isoleucine 5 of incorporation of proline into protein, its conver-

2,4-Dichlorophen- 0.5 L-Prolinie 16 sion to hydroxyproline, and the turnover ofboth pro-

oxyacetic acid line and hydroxyproline in proteins.L-Glutamic acid 51 Sucrose 20g/liter Aliquots of suspensions of proteins, homogenates,L-Aspartic acid 18 Sodium to pH 6.5 or solutions of proline and hydlroxyproline were

hydroxide counted in thedioxane-naphthalenemlixture of Butler

(1) in a Packard Liquid Scintillation Counter Modelofmediumii. Dry weights weremeasured on aliquots 314EX-2. To keel) finely divided solids in the couIlt-of fresh tissue dried at 600 for 24 to 48 hours. ingmixture from settling, 1 volunle of Cab-O-Sil, a

Cells were broken by grinding for5 to 10 minutes thixotropic gel powder, was added to each volume ofin 0.01 M Tris-0.25 Nt sucrose buffer, pH 7.5. One- counting mediuml. Proteins which would normallyhalf to 1 nml of the buffer solution was used per gram settle and escape counting now gave the results that

fresh weight of cells. Grinding was (lone in amotor- would be expected if the sample were soluble. Thedriven Thomas Teflon-glass homogenizer or a Kontes ad(lition of Cab-O-Sil lowered counting efficiency 3 to

all-glass homogenizer. Homogenates were filtered 5 % from the 51 % expected for carbon-14 or fromlthrough Miracloth to separate protoplasnm from cell- the 27 % for tritiuml. Where both carbon-14 andlwall fractions. The latter were reground, washed tritium were countecl in the same sample appropriatewith water, and refiltered to remove residlual proto- standlards were run and corrections applied.

plasm. The proteins in the filtrates wereprecipitated Hy,drolysis and Amelino Acid Assav. Protein.sby addlition of an equal volume of 40 %- trichloroacetic and cell-wall fractions vwere hydrolyzed in 6 N HCIacid. in sealed ampules at 1200 for 16 hours. The content.

Protein was assayed with bovine seruml albumin of the ampules wvere filtered, and the filtrates clrie(las standlard (10). In order to remove adsorbed a,t 1000 under a streanm of nitrogen. Proline and(amino acids before protein assay, the solutions treated hydroxyproline wvere separated by a 2-stage chroma-with trichloroacetic acidl were heated at 900 for 30 tographic procedure. First the residues were chro-minutes. Protein was precipitated by cooling the matographed downflow on NVhatman 3M.M papersolutions in an ice bath for 30 minu!tes anl collected wvith isopropanol: pyridline: water: acetic acid (8: 8:in a centrifuge at 1000 X g for 30 minutes. The pel- 4: 1). Proline-C14 and hydroxyproline-C14 wverelets were washed with 3 successive washes of cold located with a Nuclear Chicago Actigraph II \Iodel10 % trichloroacetic acid, followed by 3 successive 1036. The labeled proline and hydroxyproline areaswashes of ethanol, and 2 washes with e(qual parts of were verified by running control strips containingethanol andI ether, and then resuspended in water or these amino acids with the labeled samples. TheNaOH. Reheating the protein in fresh 20 % trich- control strips were sprayed with ninhydrin to locateloroacetic acid did not dissolve any addlitional amino the desired areas. Average RF for proline was 0.35acid. Adsorbed amino acids were removed from and for hydroxyproline 0.26. To obtain a betterwashed cell walls by heating the latter at 900 for 30 separation of these amiino acids, the combined proline-minutes in 0.2 Mf HCl. The walls were collected in hydroxyproline areas were cut from the chronma-a centrifuge and washed twice with 0.2WI HCl, twice tograms, eluted 5 timies with water, concentrate(dwith water, and then resuspended in water. undler a nitrogen streaml to about 0.2 ml, reapplied to

A pro,tein-rich fraction was extracted from washed strips of Whatnian 3MM paper, aindl chromatographe(dcell walls by grinding them 4 to 8 times in 1 N NaOH. upflow with isopropanol: 100 % fornic acid: waterThe extracts were combined and neutralized with (75: 10: 10). In this solvent the average RF forconcentrated HCl. Protein was precipitated from proline was 0.45 and for hydroxyproline 0.28. Thethe neutralized extract with 25 % ammonium sulfate, respective areas were located as before, cut separatelycollected by centrifugation, and freed of ammonium from the chronmatogranms, andl elutecl with water.sulfate by (lialysis against water. The fraction s-o Aliquots of the eluates wvere counted in a liquid

544 PLANT PHYSIOLOGY



OLSON-PROTEINS AND PLANT CELL WALLS

scintillation counter and assayed for proline andhydroxyproline according to the procedures of Trolland Lindsley (21) and Neuman and Logan (13).

When we ran measured quantities of proline andhydroxyproline through this system of analysis, 85to 90 % were recovered. The 2-stage chromatog-raphy was necessary for a good separation of prolineand hydroxyproline from each other and from otheramino acids. The results of analyses for proline andhydroxyproline by this procedure were the same as

those from a Beckman Spinco Amino Acid AnalyzerModel 120, using methods described by Spackman,Stein, and Moore (20), except in that the tempera-ture of the chromatographic column was lowered to100 to separate the hydroxyproline better.

Enzymic Digests. Labeled samples were digestedwith trypsin by incubating 1 to 2 mg samples with10 mg Worthington crystalline trypsin, 0.003 mg

sodium carbonate, and 4 ml of water at 370 for 14to 24 hours. Insoluble material was removed in a

centrifuge, aliquots of the supernatant fluid were

chromatographed directly, and the radioactivity lo-cated with an Actigraph. Other aliquots of thesupernatant fraction were hydrolyzed in 6 N HCl, thehydrolysates chromatographed as described previ-ously, and the activity of proline and hydroxyprolinedetermined. Labeled samples were also digested withStreptomyces griseus protease (California Corpora-tion for Biochemical Research Pronase, researchgrade) by incubating 50 to 200 mg of cells or cellwalls with 2 to 4 ml of a solution of 1 mg/ml proteasein 0.03 M sodium phosphate, pH 7.4, for 4 to 24 hoursat 37° (14). Aliquots of supernatant fractions were

analyzed in the same way as trypsin digests. Inaddition, aliquots of the residues were hydrolyzed in6 N HCl and the specific activities of the proline andhydroxyproline in the hydrolysate determined.

Results

Description of the System. The cultures usedwere from a line of cells that had been subculturedover 20 consecutive times. In each of these subcul-tures the same changes in morphology, chemical com-

position, and biological activity of the cells with theage of the culture were observed.

As Dougall and Shimbayashi already reported fora tobacco callus culture (2), we found that walls of

suspension cultures of tobacco cells contain a majorpart of the protein-bound amino acids of the cells.The fractionation of the cells used here is designedto differentiate between fractions on the basis of theirhydroxyproline to proline ratio and to make possiblea study of the steps involved in incorporation of pro-

line into proteins associated with cell walls and hy-droxylation of proline to hydroxyproline.

One hundred grams fresh weight of these cellsyields 7 g dry material of which 20 % is washedcell wall. About 40 % of the total protein in 10-day-old cells is found in the protoplasm and an equalamount is extractable from the cell walls. Theremaining 20 % is attached to the cell walls but isnot readily extractable. These data are summarizedin table II which includes the proline and hydroxy-proline contents and typical hydroxyproline to prolineratios of each fraction. The extractable protein frac-tion was removed from cell walls with 0.1 to 1 NNaOH, 2 M KCl, or 4 M urea. Several extractionsare necessary to remove all of the material. Thehydroxyproline to proline ratio of the extracted ma-

terial is in the range 0.7 to 1.2. Successive extractsexhibit a constant hydroxyproline to proline ratio,suggesting that the wall residual material, with a

different hydroxyproline to proline ratio, is not re-

moved by this extraction procedure. These resultsdiffer somewhat from those obtained by Dougall andShimbayashi on tobacco callus tissue (2). Theyfound that the binding of hydroxyproline-containingprotein to the cell wall resisted 6 M urea or 0.1 NNaOH at room temperature and 90 % formic acid at1000. The hydroxyproline to proline ratio of theirresidual fraction was 2 to 4, which is similar to thatreported here for residual protein. We extracted thecell-wall fraction referred to in the following para-

graphs with 1 N NaOH.The hydroxyproline to proline ratio of the pro-

tein in the buffer or wash water of the cell walls was

always in the range 0.1 to 0.2. Further, microscopicexamination of this wash revealed that it containednuclei and other protoplasmic particles and no cell-wall material.

Hydroxyproline is found in the protoplasmic, wall-extracted and wall-residual protein fractions but hasnot been found in the free state. Over 90 % of thehydroxyproline is in the 2 wall fractions. The freeproline reported in table II was found in the super-

Table II. Protein, Proline, and Hydroxyproline Content of Tobacco Cells Grouvrn on Completely Defined Medium

Mg per gram dry weight cells

Fraction Hydroxy-Protein Proline proline proline/

proline

Free ... 0.15 < 0.05Cytoplasm plus nuclear 12 0.6 0.13 02Wall extractable 12 0.4 0.3 0.8Wall residual 6 0.7 1.7 2.5

545



PLANT PHYSIOLOGY

Table III. Ammno Acid Composition ofCell-Wall Fractionts

Residues/1000

Amino acid Cell-wall Cell-wallextracted residualfraction fraction

HydroxyprolineAspartic acidThreonineSerineProline

Glutamic acidGlycineAlanineValineMethionineIsoleucine

LeucineTyrosinePhenylalanineLysineHistidineArginine

8397536866

777372736

50

823441682236

18765433880

565553897

35

6138261053923

natant of cell protoplasm separated -at 10,000 X g.In addition, cell-wall preparations which hacl notbeen washed in acid and protein fractions from theprotoplasm contain adsorbed proline which was quali-tatively identified as such by paper chromatographyof the acid washes. Assuming this adsorbedI prolinehas the same specific activity as the proline isolatedfrom the 10,000 X g supernatant, the adsorbed pro-line per gram dry weight must be several times thefree proline in the supernatant separated at 10,000 Xg.

The amino acid compositions of cell wall extractedand residual fractions (table III) are similar to thosereported by Dougall and Shimbayashi (2) for their

fractions soluble in formic acid as well as their re-i-dual fractions with the major exception of hydroxy-proline, already noted. Table III shows more histi-dine, lysine, valine, proline, and hydlroxyproline andless arginine, phenylalanine, leucine, isoleucine, ala-nine, glycine, glutamic acid, serine, threonine, andaspartic acid in the wall-residual fraction than in thewall-extracted fraction. There Nwas more threoniineancl serine in Dougall and Shimbcayashi's wall-residualfraction than in the fraction they extracted withformic acid.

The wall-extractedl fractions are only 50 c%(,, pro-tein as determined by total nitrogen analysis. IMildaci(l hydrolysis andl chromatography of the hydro-lysate shows that a nmajor portion of the nonproteinipart of these fractions is in 3 reclucing sugars, theprincipal one of which is glucose.

Incorporation of Proline-C14. We addled 30 ,ucof uniformly labele(d L-proline-C14 (S.A. 205 ,uc /nlmolefronm New Englancl Nuclear Corporation) to a slurryof 70 g of cells suspen(le(l in 70 ml of mecliumii. Afterincubation with gentle shaking for 0.5 hours at 27to 280, an aliquot of the cells was removed for frac-tionation and analysis. The specific activity of theproline in the remaining cells was (lilute(I by washingthem with fresh nmedium containing 2.0 mgmnol un-labeled L-proline and reincubating them in some ofthis nme(lium. Aliquots of the reincubatel cells vereremoved at various timles and the specific activitiesdetermined for the proline and hydroxyproline incor-porated into protein.

The drop in specific activity of protoplasmic pro-tein after removal of the original labeledl proline an(dacldition of the large excess of unlabeledl proline (fig1) indicates rapid turnover. Not all the labeled pro-line leaves this fraction rapidly, however, since thespecific activity of the protein approaches a lowerlimit.

The changes ill specific activities of proline and

Table IV. Balance Sheet of the Net Loss in Activity i it the Protoplasmt against thtc Nct Gain in Activity in theCell Walls in a Pulse-Chase Experiment of Tobacco Callnts Cells Uilsing Prolinze-C'4

TotalChange

Hproline* + proline

Protoplasmic0.5 hour pulse

After 6 hourchase

Wall extracted0.5 hour pulse

After 6 hourchase

Wall residual0.5 hour pulse

After 6 hourchase

Hproline 27,000Proline 115,000Hproline 8,000Proline 62,000

HprolineProlineHprolineProline

HprolineProlineHprolineProline

6,90031,00016,00030,000

9,10024,00061,00049,000

* Hydroxyproline.

ProteinFraction

Cpm/gdry wt Change

- 19,000- 53,000

+ 9,000- 1,00()

- 72,000

+ 8,000

+ 77,000+ 52,000+ 25,000

546




e ,

zwI.-

aci.(9

LU

Oa-

0

x

a-(0

10

6

4

0It

LABELADDED

2 4 6 8 10 12

HOURS

FIG. 1. The change in specific activity of the proteinin fractions of tobacco cells after being incubated withlabeled proline, and then chased.

hydroxyproline in the 3 types of protein during thecourse of the pulse and chase are shown in figures 2,3, and 4. The specific activity of the protein in thecell-wall extracted fraction does not rapidly declineduring the chase but first increases 20 % over itsvalue at the end of the pulse. The specific activitiesof both proline and hydroxyproline in the protoplas-mic fraction are originally high and approximatelyequal. Both decline rapidly, and appear to approachthe same lower limit. Some turnover occurs in theproline from the wall-extracted fraction but not in thehydroxyproline, at least not for a long time. Instead,the specific activity of the hydroxyproline continuesto increase long after 'the chase has begun. Theincorporation of label after the labeled substrate hasbeen removed and the medium flooded with unlabeledproline continues for 1 hour for proline and over 10hours for hydroxyproline in the wall-residual frac-tion. There is no evidence of any turnover of eitheramino acid in this fraction. The fact that proline-C14is steadily incorporated into the wall-residual fractionboth as proline-C14 and hydroxyproline-C14 long afterthe chase shows that the proline-C14 which goes intothis fraction must come from a source other thanfree proline. The data in table IV suggests thatwall protein might come from the protoplasm fraction.In this table, the change in label per gram dry weightof cells in the 3 cell fractions is tabulated for theend of .the pulse and after 6 hours of the chase. Theloss of 72,000 cpm from the protoplasm fraction isabout equal to the 85,000 cpm gained by the combinedwall fractions.

Effect of Added Proline. An important questionin these experiments is whether the metabolic proc-esses of the cells were altered by the proline in thepulse and the large excess of proline in the chase.

Less than 0.1 ,ug of proline-C14 per gram of incuba-tion medium was necessary to assure sufficient incor-porated label for counting purposes. This is con-siderably lower than the concentration of free prolinein either the cells or medium. Hence we can assumethat the cells are unaltered by the addition of the pulse.

The amount of the chase, however, was necessarilymuch larger. To study the effect of the chase, analiquot of cells was removed from the reaction flask2 hours after the chase, washed with normal medium,and reincubated with labeled proline. Under theseconditions *the cells incorporated labeled proline intoall protein fractions, which shows that the cells werenot seriously damaged by the chase and that proteinsynthesis continued.

There was the possibility that a metabolic processmight be reversibly altered by the presence of a largeexcess of proline in the medium. If this is true,removal of the excess proline in the medium wouldthen restore the original biochemistry. To checkthis, 13 g of cells were incubated for 0.5 hours with10 ,uc of L-proline-C14 and 50 ,uc of L-lysine-4,5-H3(4.1 c/mmole, New England Nuclear Corporation) in78 ml of medium. Enough unlabeled L-proline wasadded to yield a final concentration in the medium of5.3 mg/ml or a dilution of -the labeled proline of morethan 100,000-fold. Under these conditions the in-corporation of lysine-4,5-H3 continued in all proteinfractions, though somewhat more slowly. Thus pro-tein biosynthesis continues even after the addition ofa large excess of proline to the medium. In thisexperiment proline incorporation followed the samepattern described for the first experiment. The dataare shown graphically in figure 5 for the protoplasmicfraction.

Effectiveness of Dilution. It was important todetermine whether the chase thoroughly diluted thespecific activity of free pools of proline in the cells.If it were not effective, then the labeled proline in-c3rporated into cell-wall protein during the chasecould come from this source. A plot of the specificactivity of the free proline for the pulse-chase experi-ment described in the preceding paragraph is shownin figure 6. The data show that the specific activityof the proline inside the cell changes very rapidlywith changes in the activity of the proline in themedium. The rapid decrease in specific activity ofthe free proline in the cell after addition of the chasemakes it clear that the label incorporated into cellwall during the chase did not come from this source.

Enzymic Digests. The wall-extracted, protein-rich fraction from cells which had been incubatedwith uniformly labeled proline-C14 was hydrolyzedwith trypsin to yield a mixture of peptides. Hydro-lysis rendered over 90 % of the radioactivity of theprotein chromatographically mobile. Radioactivityappeared in more than 5 peptides with less than 5 %in free proline or hydroxyproline. Acid hydrolysis ofthese peptides and chromatographic separation of theamino acids showed that the peptides contained bothproline-C14 and hydroxyproline-C'4. Trypsin does

WALL EXTRACTED FRACTION

rHi .,

O.5 HOURi

r] APu-

547

8



PLANT PHYSIOLOGY

PROTOPLASMICFRACTION

PROLINE\3 HYDROXYPROLINf-------.,0HYDROXYPROLINE

CHASE0.5 HOUR

IlI5 10 15 20 25

HOURS

0 5 10 15t HOURS

LABELADDED

FIGS. 2, 3, 4. The change in specific activities of the proline and hydroxyproline in the protoplasmic (fig. 2, upper

left), wall-extracted (fig. 3, lower left), and wall-residual (fig. 4, upper right) fractions of tobacco cells after thelabeled proline in which they were incubated was diluted with a large excess of unlabeled proline. Dilution of ex-

ogenous C14-labeled proline with unlabeled proline in excess of 10,000: 1.

not attack cell-wall residual protein under the condi-tions of the hydrolysis used.

Proline-C'4 labeled whole cells and cell-wall prep-

arations enzymically hydrolyzed with Streptomycesgriseus protease also gave a mixture of peptides. Thehydroxyproline/proline ratio in these peptides is in therange 0.2 to 1.0, characteristic of protoplasmic andwall-extracted protein. After treatment with thisprotease, the hydroxyproline/proline ratios in wholecells and cell-wall preparations were in the range 2to 4, characteristic of residual wall protein. Residualcell-wall protein was not attacked by Streptomycesgriseus protease.

Discussion

Steward et al. have reported that bound hydrox-yproline is an end product of metabolism in culturedcarrot cells and potato disks and that proline is con-

verted to hydroxyproline after the proline has beenbound, presumably into protein (11, 16, 19). Man-ner and Gould (12) working with chick embryoshave presented results which suggest proline is hy-droxylated prior to its assembly into collagen. Thesedata support the hypothesis that there exists a poolof activated proline which can be hydroxylated togive activated hydroxyproline which is subsequently

548

aC

40z

4w-j0

E

w

0-

25

a

204 20.0z

< 15w-J0

510

w

a.)

0

LAEADC

a

40z

4w-402

E

w0-

0.

ELIED

20 25

lu




z

20

(L ast \PROLINE-C14 (al.)

16

w 0

ito10

x LYSINE-4E5-H3

4

PRIOLINE CHASE

05 2 3 4 5 6

LABEL HOURS

ADDEDFIG. 5. The effect of a proline chase on the incorpora-

tion of uniformly labeled proline-C14 and lysine-4,5-H3into protoplasmic protein.

W 1,000z

0

a: 800 1a.

W

W

Lh- 600W

-J

0

400

E

W 200a.

a.

0 0.5 4 5 6

LAt HOURS

ADDED CHASE

FIG. 6. The change in specific activity of the freeproline in the cells in a pulse-chase experiment.

incorporated into collagen. The fact that they were

able to isolate s-RNA-proline and s-RNA-hydroxy-proline complexes led these investigators to suggestthat such complexes may be the active compoundsinvolved in hydroxylation and collagen biosynthesis.On the other hand, Peterkofsky and Udenfriend (15)working with cell-free systems from chick embryohomogenates have suggested that the substrate forhydroxylation is a microsomal RNA-bound polypep-tide of considerable size. From studies with spongebiopsy connective tissue, Kao et al. (5) have pre-sented data which suggest that all collagen hydro-xyproline is derived from proline peptide.

We have shown that in pulse-chase labeling ex-

periments with cultured tobacco cells, proline-C14appears as proline-C14 and hydroxyproline-C14 firstin protoplasmic protein and later in cell-wall asso-ciated proteins. The loss of labeled proline andhydroxyproline from the protoplasmic protein withtime is about equal to the gain of these labeled aminoacids by the wall protein. These results suggest thathydroxylation of proline to hydroxyproline occursafter proline is incorporated into a protoplasmic pro-tein or polypeptide and before or during its depositionin the cell walls. The results agree with the workof Steward et al. (19), Peterkofsky and Udenfriend(15), and Kao et al. (5) but do not rule out thepossibility that hydroxylation occurs on an activatedproline. Such an intermediate might have been lostor destroyed during our method of fractionating thecells, or it might have been adsorbed on proteins orother polymers and destroyed by the trichloroaceticacid treatment. If, however, the large amount oflabel incorporated into cell-wall protein after thechase, mainly as hydroxyproline, were all in a solu-ble active form at the beginning of the chase it shouldhave been possible to isolate this active species. Wewere not able to isolate an active proline by chroma-tography of the 10,000 X g supernatant fractions.

By radioautographic techniques we have been ableto tentatively confirm that the hydroxyproline-richproteins are associated with cell walls. Protoplasmicproteins, but not hydroxyproline-rich ones, are re-moved from whole cells by enzymic hydrolysis.Radioautographs of hydrolyzed cells previously la-beled with proline-C14 show localization of label inthe area of the cell wall.

The resistance to separation from the wall indi-cates that cell-wall-bound amino aci'ds may be cova-lently bonded to the wall. Lamport (6, 7) hassuggested covalent bonding to hemicelluloses or cel-lulose. Failure of proteolytic hydrolyzing enzymesto release peptides or amino acids from the cell wallssupports this hypothesis.

Summary

The proteins of tobacco cells grown in suspensionculture on a completely defined medium have beenfractionated into a protoplasmic fraction, a cell-wall-extracted fraction, and a cell-wall-residual fraction.Each fraction is described in terms of separation fromthe cell, hydroxyproline to proline ratio, amino acidcomposition, and susceptibility to enzymic hydrolysis.The hydroxyproline to proline ratios for the 3 frac-tions are 0.2, 0.7 to 1.2, and 2 to 4, respectively.

In vivo pulse-chase experiments with proline-C14have shown that this amino acid is very rapidly in-corporated into protoplasmic protein, more slowlyinto cell-wall-extracted protein, and much more slowlyinto residual cell-wall protein. A portion of theprotoplasmic protein turns over very rapidly, whereasthe cell-wall proteins do not turn over rapidly, if atall. The turnover in protoplasmic protein may berelated to the incorporation into cell-wall protein.

549



PLANT PHYSIOLOGY

Acknowledgments

The author wishes to thank Professor James Bonnerfor his suggestions and encouragement, Miss Joyce Bul-lock for the amino acid analyses, and Mr. Bruce Bowenfor his techniical assistance.

Literature Cited

1. BUTLER, F. E. 1961. Detcrmination of tritium inwater and urine-liquid scintillation counting andrate-of-drift determination. Anal. Chem. 33: 409-14.

2. DOUGALL, D. K. AND K. SIIIMBAYASHI. 1960. Fac-tors affecting growth of tobacco callus tissue andits incorporationi of tyrosine. Plant. Physiol. 35:396-404.

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proteins and plant cell walls. proline to hydroxyproline ... · determination h3bo3 1.5 glycine 5...

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