May 22, 1972 - The radioactivity of soluble radioactive samples, diluted to 1 ml with water, was counted in 10 ml of toluene-Triton X-100 (2:1 v/v) scintillant (22).
Plant Physiol. (1972) 50, 531-535
Location of Glycoproteins That Contain Glucosamine in Plant Tissues1 Received for publication May 22, 1972
R. M. ROBERTS,2 J. J. CETORELLI, E. G. KIRBY, AND MARY ERICSON Departments of Biochemistry and Botany, University of Florida, Gainesville, Florida 32601, and Department of Biology, University of California, San Diego, La Jolla, California 92037
surface-sterilized with 1.25% (w/v) sodium hypochlorite solution for 15 min, washed with sterile water, and germinated on When radioactive D-glucosamine is provided to Acer pseu- 1% (w/v) agar at 25 C in darkness. doplatanus cells in liquid culture in order to label those glycoSycamore (Acer pseudoplatanus) callus was maintained on proteins that contain amino sugars, it is incorporated pre- the defined agar medium containing sucrose, originally deobservation fraction. This was crude wall a cell dominantly into scribed by Murashige and Skoog (13). Pieces of this callus (2 confirmed histologically by preparing autoradiographs of thin to 3 g) were transferred aseptically to 100 ml of freshly pretissue sections from plasmolyzed cells. Highly purified cell wall pared liquid medium and held at 28 C on a gyratory shaker material from unlabeled cells has also been shown to contain (250 rpm) for about 20 days. At this stage 10-ml samples (apsmall amounts of glucosamine. Similarly, about one-half of the proximately 4 ml packed cell volume) of the cell suspension amino sugar recovered from cultured cells of Nicotiana which resulted were pipetted into several new flasks, each contabacumn is present in their cell walls. In corn roots, however, taining 90 ml of fresh medium. Growth was then allowed to the labeled glycoproteins that are formed after glucosamine continue and cells were transferred routinely every 2 weeks. incorporation are predominantly cytoplasmic and not deposited The growth curve shown in Figure 1 is reproducible under outside the protoplast. these conditions. Cell division figures were confined to the first 5 days in new medium (E. G. Kirby, unpublished results) and all subsequent growth was the result of cell enlargement. Tobacco callus cells (Nicotiana tabacum) were grown in 300 ml of MI-D liquid medium (5). Each flask was inoculated with to 0.4 g of cells from a 6-day-old culture and harvested D-Glucosamine is a widespread, although relatively minor, 0.3 as required. component of plants. It is usually found in combined form as a (specific radioactivity 53 mc/mmole) constituent of glycoproteins which have included a number of andD-Glucosamine-1-'4C D-glucosamine-1-'H (specific radioactivity 2.6 c/mmole) enzymes (14, 19, 20) and such potentially interesting com- were obtained from Amersham-Searle Corporation, Arlington pounds as the phytohemagglutinins (7, 11, 21). Little is known, Heights, Illinois. These were prior to the experiments however, about the cellular location of glycoproteins in many described here and shown toanalyzed be more than 98% radiochemhas that living tissues, although Eylar (4) proposed glycosyla- ically pure. Crystalline trypsin was a product of Worthington tion of proteins may, in some undefined way, be necessary for Biochemical Corporation, Freehold, New Jersey, Triton X-100 their secretion and that glycoproteins are, therefore, predomi- of the Rohm and Haas Company. All other chemicals were of nantly extracellular. Certainly a few secreted proteins that have the highest purity obtainable. cells do have from bound carbobeen analyzed plant covalently Labeling Experiments. At the time of transfer, Acer cells hydrate attached to them, but the status of others is unclear and were provided with D-glucosamine-1-"4C (10 ,uc/100 ml) or it is to label information is generally sparse. However, possible D-glucosamine-1-'H (100 jic/100 ml) in the standard medium those plant (17) and animal glycoproteins (9) that contain under aseptic conditions. Corn roots (40) were shaken (100 amino sugars by supplying the tissues with radioactive D-glu- rpm) with D-glucosamine-1-2H (100 ,uc) in 5 ml of distilled cosamine. By autoradiography it should then be possible to water for 4 hr at 30 C. determine where these glycoproteins are to be found. AlternaAnalytical Procedures. To release the amino sugars from tively, the cells can be broken open and the distribution of cell residues, portions the material were hydrolyzed with 4 glucosamine into cell wall and intracellular components esti- N HCl at 105 C for 4 hrof(or 1 N HCl for 24 hr) in a sealed tube mated chemically. The results of such autoradiographic and under nitrogen. The suspension was then centrifuged and the chemical studies are reported here for a number of plant tis- supernatant solution dried in vacuo to remove HCl. Portions of sues. this were then chromatographed for 40 hr in the following solvents: (solvent A) 1-butanol-ethyl acetate-acetic acid-water MATERIALS AND METHODS (5:5:3:1 v/v) in a tank equilibrated with pyridine-ethyl aceMaterials. Corn (Zea mays var. Golden Bantam) was pur- tate-water (11:40:6 v/v); (solvent B) 1-butanol-pyridine-0. 1 chased from Burpee's Seeds, Sanford, Florida. Seeds were N HCI (5:3:2 v/v). Either chromatograms were scanned for 4C using a Packard 7201 scanner or autoradiographs were with Kodak Industrial x-ray film. I prepared Supported by National Science Foundation Grants GB 23533 To hydrolyze proteins for total amino acid analysis, cell (to R.M.R.) and GB 30235 (to M. J. Chrispeels). residues were heated with 6 N HCl at 105 C for 16 hr in a 2To whom reprint requests should be addressed. ABSTRACT
531
532
ROBERTS, CETORELLI, KIRBY, AND ERICSON
sealed tube under N2. The acid in the supernatant fraction was removed by evaporation and the hydrolysate dissolved in 0.1 N HCl. Amino acids and amino sugars were analyzed on a Beckman model 120 C Amino Acid Analyzer, using a program recommended by the manufacturers (Technical Bulletin A-IM-3, June 1965). However, in order to separate hydroxyproline from aspartic acid, the sample was introduced to the long column using 0.2 M citrate buffer at pH 3.20 rather than pH 3.28. Under these conditions hydroxyproline is eluted approximately 5 min ahead of aspartic acid. Amino sugars and basic amino acids were eluted from the short column in the usual manner using 0.35 M buffer at pH 5.28. In the experiments with tobacco, amino sugars were determined quantitatively by a modification of the Elson-Morgan method (1) in which amino sugars are separated from interfering amino acids on a short column of Dowex 50 (H+) cation exchange resin. Determination of Radioactivity. The radioactivity of soluble radioactive samples, diluted to 1 ml with water, was counted in 10 ml of toluene-Triton X-100 (2:1 v/v) scintillant (22). Insoluble residues were counted as a suspension in the thixotropic gel, Cab-o-sil (Packard Instrument Co., Downers Grove, Ill.). Analysis of Radioactive Acer Cells. Duplicate samples of Acer cells were removed from the culture medium using wide bore pipettes, centrifuged in graduated centrifuge tubes, and their packed volumes measured. They were washed with water and then ground up in 80% (v/v) ethanol using a ground glass, motor-driven homogenizer. After being centrifuged, the residue was extracted several times with ethanol until the washings were free of '4C, dehydrated using 100% ethanol and then ether, and allowed to air-dry. The 80% ethanol extracts and
Plant Physiol. Vol. 50, 1972
washes were pooled and referred to as the ethanol-soluble fraction. The ethanol-insoluble residue was then extracted four times with 0.5 M NaCl containing 0.1 S% Triton X-100. Samples of the final residual material counted as a suspension in Cab-o-sil. Isolation of Cell Wail from Acer Cells. Cells from 14-dayold cultures were cooled to 0 C and broken open by sonication (Bronwell, Biosonic at full power with 3/4-inch probe) for 20 min in presence of 0.5 M sodium chloride containing 0.1 % Triton X-100. The fragments were then finely ground using a ground glass Potter homogenizer. The pellet which was collected by centrifugation at 500g was washed four times with fresh grinding medium. At this stage we could detect no unbroken cells by microscopic examination, but could not ensure that the pellet was uncontaminated by cytoplasmic debris. Portions of this material, therefore, were suspended in glycerol (specific gravity 1.26) and the dense cell wall collected by centrifugation (lOOOg; 10 min). This procedure was repeated four times. The final residue was washed with water to remove glycerol, dehydrated, dried, and divided into two equal portions. One of these was acid-hydrolyzed while the other was suspended in a solution of trypsin (0.05% w/v) in 50 fM NHJICO3 at 37 C. After 24 hr the trypsin-treated residue was washed with water, dehydrated. and dried prior to hydrolysis. Analysis of Nic tiany Cells. Cells were collected by centrifugation and ground up in ice-cold distilled water using a conical, all glass homogenizer. The crude cell wall fraction was centrifuged (1 500g; 5 min), washed four times with water, once with ethanol, and air-dried prior to hydrolysis. Cytoplasmic proteins were precipitated from the supernatant fraction of the ground cells by addition of trichloroacetic acid (to 7.5% w/v). The precipitate was centrifuged, washed once with 5% (w/v) trichloroacetic acid. twice with ethanol, dried, and
hydrolyzed. Histological Procedures. Approximately 4 ml of the cell suspension were removed from the Acer culttures. These cells were plasmolyzed by placing them in 1 ml of 5 M sucrose for 30 min. They were then fixed in 2.5% glutaraldehyde in 50 mM cacodylate buffer, pH 7.2 (30 min), postfixed in 2.0% osmium tetroxide which had been prepared in the same buffer (90 min), washed, and the pellet embedded in 1% (w/v) agar. The agar block was then dehydrated and embedded in Araldite plastic (3). Sections (0.4 yrm) were cut with a Porter-Blum MT-2 ultramicrotome using glass knives and were mounted on slides in 0.1 % (w/v) sodium borate. Root tips were fixed in 6.12% glutaraldehyde in 50 mM cacodylate buffer at pH 7.4, washed, and postfixed as above. They were then dehydrated in an ethanol-acetone series and embedded for sectioning in Epon-Araldite plastic (12). Autoradiographs were prepared by dipping the slides in Kodak NTB-2 nuclear track emulsion diluted 1:2 with distilled water. The emulsion was developed in Kodak D-19 developer for 2 min, washed, and fixed for 5 min. Sections were treated with 0.1% w/v aqueous toluidine blue which stains nuclei and cytoplasmic constituents, but not the cell walls.
0
I
E
a -Dc
n
5 4 °-_
2-
CXD
3
-
3
a~
=Z=-,I±w 2
6
4
8
2
1-
1
P
_-AAQ 10
12
16
14
RESULTS AND DISCUSSION
Days FIG.
1.
growth
in
cosamine 10-ml
Incorporation liquid culture. in
100
samples
12, 14,
and 17.
ml
of
D-glucosamine
Cells were
of medium at the
were removed from
14C in
(A),
wall) fraction (A), and in the medium are expressed as the average of the two cell volumes
(0)
for 10-ml
samples
,uc
with 10
time of
duplicate
the ethanol-soluble
soluble-ethanol-insoluble fraction
into A cer cells
provided
of
during D-glu-
transfer, and single days 3, 5, 10,
flasks at
fraction
(A),
in the NaCl-
in the NaCl-insoluble
(A) is 10-ml
(cell
indicated. All results
samples.
The
of medium are also
packed
shown.
Incorporation of Glucosamine in Acer Cells. When D-glucosamine-'4C was supplied in the growth medium of Acer cells, it was not taken up immediately, but was gradually accumulated by the cells over the entire growth period (Fig. 1). Net uptake was completed around day 14, at which stage the cul-
ture mass had become almost constant. At whatever stage the cultures were sampled, a large portion (usually around 60 to 70%) of the "4C was recovered in ethanol-soluble materials of low molecular weight. In the experiments reported here, this
Plant Physiol. Vol.
50, 1972
P
GLYCOPROTEIN LOCATION
533
FIG. 2. Autoradiographs of sections from plasmolyzed Acer cells which had been provided with D-glucosamine-3H for 5 days after transfer. Both sections are 0.4 ,um thick and stained with toluidine blue.
fraction was discarded. Of the remaining ethanol-insoluble radioactivity, only a small part could be extracted by exhaustive treatment with 0.5 M NaCl containing 0.1 S% Triton X-100. The rest seemed firmly bound to cell residue, and, because it was so insoluble, it seemed possible that it was associated with the cell wall. It is curious that Acer callus grown on solid medium incorporated relatively less 14C into NaCl-insoluble macromolecules and relatively more into soluble glycoproteins than the suspension cultures used in these studies (17). We have no explanation for this difference. To test whether the 14C was present in amino sugars, a portion of this final residue was hydrolyzed (4 N HCl; 4 hr; 100 C) and chromatographed in solvents A and B. Seventy per cent of the radioactivity was associated with glucosamine, while the rest remained at the origin, presumably in the form of partial hydrolysis products (17). Galactosamine was not present in the hydrolysate while the commoner monosaccharide components of the wall and amino acids were unlabeled. Autoradiography of Acer Cells. We confirmed that radioactivity was present in the wall by preparing autoradiographs of sectioned material. Cells were grown on media containing D-glucosamine-3H. Samples of cells were removed from the suspension cultures 3, 5, 7, 9, and 14 days after transfer to the radioactive medium and plasmolyzed prior to fixation (18). We found this to be necessary in order to separate the cytoplasm from the cell wall. Acer cells, like others in culture, are highly vacuolate even during the meristematic phase of growth (up to day 5), and it is difficult, even on thin sections (0.4 um) of unplasmolyzed cells, to distinguish unequivocally between label in the cytoplasm and that in the wall. The autoradiographs indicated that a large proportion (over 70%) of the 'H was present in the cell walls at all the stages we examined (Fig. 2). By contrast, silver grains were relatively few over the clumps of cytoplasm that were visible on some of the sections. Radioactivity seemed to be distributed evenly over the wall and was not apparently localized in any one region. Analysis of the Acer Cell Wail. The occurrence of glucosamine as a normal constituent of the cell wall rather than as a component introduced during the labeling experiment was shown when we analyzed 14-day-old tissues grown in medium that did not contain the amino sugar.
Table I. Aminzo Acid Composition of Insoluble Residutes of Acer Cells In each case the starting material was 50 mg of salt-extracted residues. Tryptophan was not estimated. Amino Acid
Salt-extracted Residue
Glycerol-treated Residue
lAmoles/
11moles/ 50 mg
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alar1ine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxyproline Glucosamine
3.7 0O.8 1.2 4.3 2.4 4.7 4.2 3.5
3.8 3.3 0.1 3.4 0.2 1.8 3.2 0.8 1.6 3.9 0.3
Trypsin-treated Residue
pmoles/
pmoles/
100 pAmo!esl jAmolesl jAmole Mtmoles 50 mg amno 50 mg amino 100
acid 7.8 1.8
2.6 9.1 5.0 9.9 8.9 7.4 8.0 7.0 0.2 7.3 0.3 3.9 6.8 1.7 3.4 8.2 0.6
2.9 0.8 0.8 2.3 1.1 2.6 2.6 2.4 2.0 1.8
acid 10.0 2.6 2.9 7.7 3.9 8.9 9.0 8.2 6.7 6.2
1.2 0.1 1.2 1.9 0.8 1.0 2.7 0.2
7.6 0.2 4.1 6.3 2.7 3.5 9.1 0.7
100
JAmolies amino
1.1
acid 10.4 2.7 1.9 5.8 3.5 9.3 6.7 8.7 6.2 5.1
1.5 0.1 0.7 1.1 0.6 0.5 3.5 0.2
9.1 0.3 3.3 5.6 2.8 2.4 15.9 0.9
2.3 0.6 0.4 1.3 0.8 2.6 1.5
1.9 1.4
Total amino acids and amino sugars were analyzed in the washed insoluble residue obtained after breaking open the cells in presence of NaCl and Triton X-100, in the residue prepared after glycerol treatment, and in the final trypsin-treated material (Table I). An analysis of cell walls at stage 1 revealed the presence of all of the commoner amino acids normally found in protein, plus an appreciable amount of hydroxyproline and the amino sugar glucosamine. Note that while the purification procedure (stages 2 and 3) reduced the net amount of the amino
534
ROBERTS, CETORELLI, ]KIRBY, AND ERICSON
acids most commonly found in protein, the content of hydroxyproline and the amino sugar glucosamine remained fairly constant. In the final preparation there was approximately 1 molecule of glucosamine for about 16 molecules of hydroxyproline. The composition of these walls is not unlike those reported by others, except for the presence of the small amount of amino sugar (6, 10). It seems clear, therefore, from these sets of observations that D-glucosamine is a component of the cell walls of Acer cells grown in suspension cultures. We still do not know into
what sort of macromolecules the glucosamine is incorporated, except that they are highly insoluble, and we have no evidence that glucosamine forms part of the hydroxyproline-rich glycoproteins of Acer cell walls. Indeed, other investigators have not indicated the presence of amino sugars in glycopeptides containing hydroxyproline isolated after partial degradation of cell walls of callus cells (6, 10). Analysis of Nicotiana Cells. The total amino sugar content of tobacco cells in liquid culture was followed over a period of Table III. Amino Sugar Content of Cell Wall and Cytoplasm of
Table II. Aminio Sugar Content of Ethanzoi-inisoluble Materials from Tobacco Cells Grown in Culture Days after Transfer
Weight of Cells
Plant Physiol. Vol. 50, 1972
Cultured Tobacco Cells Time after
Glucosamine Content
Transfer'
Glucosamine
WVeight of Cells In wall
0 3 6 9 12
g
total pug
;,g/g freshi wt
0.40 0.95 1.10 2.40 6.50
17 34 44 81 245
42 36 40 34 38
A
days
g
3 6 9
0.97 2.00 2.90
In cytoplasm plg
19.8 36.0 43.5
24.3 36.4 47.0
Inoculum size was 0.3 g/flask.
B
C
*l
A
D
E
FIG. 3. Autoradiographs of sections from corn roots provided with D-glucosamine-3H. A: 4 ,m median longitudinal section (Ls) of root-tip x 50 photographed under dark ground illumination; B: 0.4 ,m Ls of junction of root-cap (lower part of section) and root proper (upper part of section) X 1000. The cell wall separating the root-cap from the root is appreciably thickened. C: 0.4 ,um Ls of the epidermis and epidermal wall (W) 1.5 mm above the root-tip X 1000; D: 0.4,um Ls of peripheral cells of the root-cap and epidermal cells 0.5 mm above tip X 800; E: 0.4 ,um Ls of root-cap cells (lower part of section) and young epidermal cells (upper part of section) showing the early thickening of the outer epidermal wall which is unlabeled X 800. Sections B-D are photographed under normal illumination and stained with toluidine biue.
Plant Physiol. Vol. 50, 1972
GLYCOPROTEIN LOCATION
535
12 days after transfer to new medium. From Table II it can be necessarily a prelude to secretion, as has been suggested, but seen that although the amino sugar content of the total culture that many of the cytoplasmic proteins may bear a carbohyincreased with growth, the amount per g of tissue remained al- drate moiety whose function remains unknown. most constant. In a separate experiment, we isolated a cell wall Acknowledgments-The authors thank Dr. R. G. Stanley for providing the fraction and a trichloroacetic acid-precipitable fraction from initial cultures of Acer cells, Doctors H. C. Aldrich and A. R. Stevens for the cytoplasm. The amino sugar was distributed about equally help with the histology, and Dr. M. J. Chrispeels for helpful discussion of the between these two fractions (Table III). It is apparent, there- results. fore, that amino sugar is present in these tobacco cell walls, LITERATURE CITED although it is again recovered in very small amounts. As in Acer, it is not clear whether the cytoplasmic material is a pre- 1. BOAS, N. 1953. 'Method for the determination of hexosamines in tissues. J. cursor of that in the wall. Biol. Chem. 204: 553-563. Incorporation of D-Glucosamine-3H into Zea Roots. Auto- 2. CLOWES, F. A. L. 1958. Protein synthesis in root meristems. J. Exp. Bot. 1: 249-260. radiographs of thin (0.4 ,um), median sections from roots fed H. D. 1967. Rapid and improved methods for embedding biological D-glucosamine-3H are shown in Figure 3, B to D. Silver grains 3. COULTER, tissues in Epon 812 and Araldite 502. J. Ultrastruct. Res. 20: 346-351. are scattered over the cytoplasm in all cells in all of the tissues 4. EYLAR, E. H. 1966. On the biological role of glycoproteins. J. Theor. Biol. 10: we have examined and are not localized close to or over the 89-113. cell walls. This can be best appreciated in cells where the wall 5. FILNXER, P. 1965. Semiconservative replication of DNA in a higher plant cell. Exp. Cell Res. 39: 33-39. is thickened. Epidermal cells between 0.2 and 3 mm from the 6. HEATH, .M. F. AND D. H. NORTHCOTE. 1971. Glycoprotein of the wall of apical zone of the root, for example, have extremely thick sycamore tissue culture cells. Biochem. J. 125: 953-961. walls (10 to 15 lum) which do not stain with toluidine blue (Fig. 7. KALB, A. J. 1968. The separation of three L-fucose-binding proteins of Lotus tetragonolobus. Biochim. Biophys. Acta 168: 532-536. 3, C, D, E). It can be seen that silver grains are relatively E. G. AND R. M. ROBERTS. 1971. The localized incorporation of 3H-Lsparse over these walls, although they are known to incorpo- 8. KIRBY, fucose into cell wall polysaccharides of the cap and epidermis of corn roots. rate polysaccharide at an extremely high rate compared to Planta 99: 211-221. other regions of the root tip (8, 16). 9. KOHN, P., R. J. WINZLER, AND R. C. HOFFMANN. 1962. Mletabolism of Dglucosamine in the intact rat. J. Biol. Chem. 237: 304-308. The section shown in Figure 2A has been photographed D. T. A. 1969. The isolation and partial characterization of hyunder dark ground illumination and at a lower magnification 10. LANIPORT, droxyproline rich glycopeptides obtained by enzymic degradation of primary than the other sections. Silver grains (and other dense particles) cell walls. Biochemistry 8: 1155-1163. appear white against a dark background. The micrograph effec- 11. LIENER, I. E. 1964. Seed hemagglutinins. Econ. Bot. 18: 27-33. tively illustrates the general pattern of incorporation within the 12. 'MOLLENHATER, H. H. 1963. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39: 111-114. first 0.6 mm of the root tip. Incorporation is clearly high in the 13. IMURASHIGE, T. AN-D F. SKOOG. 1962. A revised medium for rapid growtth and cells on the margins of the root cap which are becoming debioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. tached. This is also illustrated at higher magnification in Figure 14. OTA, T., S. MOORE, AND WV. H. STEIN. 1964. Preparation and chemical properties of purified stem and fruit bromelains. Biochemistry 3: 180-185. 2D. The quiescent center which is visible as a darker, cupR. M. 1970. The incorporation of D-glucosamine-14C into root tisshaped area in the center of the section stands out as a region 15. ROBERTS, sues of higher plants. Plant Physiol. 45: 263-267. band of active cells below 16. ROBERTS, R. 'M. AND V. S. BUTT. 1968. Patterns of incorporation of pentose of low incorporation, while the thin it corresponds with the root cap initial cells. Incorporation is and uronic acid into the cell walls of maize root tips. Exp. Cell Res. 51: 519-530. low in the central regions of the root (central pith) but rela17. ROBERTS, R. 'M., A. B. CONNOR, AND J. J. CETORELLI. 1971. The formation of tively high in the outer cortex and epidermis. glycoproteins in tissues of higher plants. Specific labelling w-ith D-[1-14C]It is apparent from these observations that in corn roots the glucosamine. Biochem. J. 125: 999-1008. incorporated radioactivity is mainly cytoplasmic and does not 18. SADAVA, D. .AND 'M. J. CHRISPEELS. 1969. Cell wall protein in plant-s: autoradiographic evidence. Science 165: 299-300. form part of the extracellular wall. This had been suspected in L. M., E. KAY, AN-D J. L. LEW. 1966. Peroxidase isozymes from earlier experiments (15, 17) when it was shown that glucosa- 19. SHANNON, horseradish roots. Isolation and physical properties. J. Biol. Chem. 241: mine was incorporated largely into glycoproteins that were 2166-2172. soluble in water and dilute solutions of salt. The pattern of 20. STARK, G. R. AND C. R. DAWSON. 1962. On the accessibility of sulfhydryl groups in ascorbic acid oxidase. J. Biol. Chem. 237: 712-716. labeling observed on autoradiographs is quite similar to that T., P. RAMACHANDRAMURTHY, AN-D I. E. LIENER. 1967. Some observed by Clowes (2) when he followed L-leucine incorpora- 21. TAKAHASHI, physical and chemical properties of a phytohemagglutinin isolated from tion to determine the relative rates of total protein synthesis in Phaseolus vulgaris. Biochim. Biophys. Acta 133: 123-133. different regions of the corn root tip. One possible implication 22. TURN-ER, J. C. 1968. Triton X-100 scintillant for carbon-14 labeled materials. Int. J. Appl. Radiat. Isotop. 19: 557-563. of these observations, therefore, is that glycosylation is not
