- Email: [email protected]
Glycoprotein biosynthesis: The solubilization of glycosyl transferases from membranes of hela cells and bovine submaxillary glycoproteins
ARCHIVES
OF
BIOCHEMISTRY
Glycoprotein
AND
BIOPHYSICS
Biosynthesis: from
447-455 (1969)
12%
The Solubilization
Membranes
of Hela
Submaxillary ARPI
Received
June
Cells
and
Glycoproteins”
HAGOPIAN
The Salk Institute
of Glycosyl
AND
for Biological
Bovine 2
E. H. EYLAR
Studies,
10, 1968; accepted
Transferases
San Diego, October
California
21, 1968
Two glycosyl transferases, the polypeptidyl:N-acetylgalactosaminyl and glycoprotein:galactosyl transferase, which participate in the assembly of the carbohydrate units of glycoproteins, were purified from both HeLa cells and submaxillary glands. In the case of HeLa cells these enzymes are intimately associated with the smooth internal membranes where they form part of the multienzyme group of transferases synthesizing membrane glycoproteins; in submaxillary tissues, however, these enzymes are involved in synthesis of secreted submaxillary glycoproteins and are strongly bound to membranes, possibly plasma membranes, which sediment, to the 3&45yo sucrose interface. In preparing the respective membranes, the glycosyl transferases were purified from l&40-fold over the cellular homogenate. It was found that from SO-95% of the total enzyme activities are masked in the isolated membranes; disruption of the membranous structure with nonionic detergents or with phospholipase A liberated the transferases. The largest increase in activity was obtained with the polyoxyethylene octylphenol type. The linear alcohol polyoxyethylene types were slightly less effective; less than 50% of the maximum activity was obtained. These results, together with the effect of phospholipase A, emphasize the significance of nonpolar forces in membrane substructure, particularly with regard to the interaction of the transferases with their membranous environment. By contrast, negatively charged detergents, such as sodium deoxycholate and sodium dodecyl sulfate, led to strong inhibition of enzymes. A positively charged detergent, cetylpyridinium bromide, also produced strong inhibition. It was concluded that the influence of detergents on the activity of membrane-bound transferases appears to depend on two factors, solubilization and inhibition.
In Dhe biosynthesis of glycoproteins, t’he steps leading to synthesis of polypeptide chain and formation of t’he carbohydrate 1 This study was supported, in part, by a grant from The National Foundation to Dr. Jonas Salk, and in part, by the American Cancer Society, California Division. 2 The terminology used in this report in referring to individual glycosyl transferases designates the macromolecular receptor and the monosaccharide being transferred, e.g., polypeptidyl:X-acetylgalactosaminyl transferase. The following abbreviations were used: galNAc, Nacetylgalactosamine; gal, galactose; SDS, sodium dodecyl sulfate; DOC, sodium deoxycholate; BSM, bovine submaxillary glycoprotein; UDP, uridine diphosphate. 447
units and the integration of these two processes must be considered. Present data (l-7) suggest that the synthesis of the polypeptide is carried out at the polyribosomes according to current concepts for simple proteins. Controversy still exists over the role of microsomes or ribosomes as a site for attachment of the carbohydrate moiety to the protein (5, 6,s). A multienzyme group of transferases involved in the biosynthesis of carbohydrate units of membrane glycoproteins has been found (9). It is reasonable to assume that the cellular location of the transfcrases dictates the site of attachment of monosaccharides to the polypeptide chain. In an earlier study with HeLa cells
44s
HAGOPIAN
it was shown that two enzymes involved in biosynthesis of membrane glycoproteins, the polypeptidyl: N-acetylgalactosaminyl and glycoprotein: galactosyl transferases are located in the same internal smooth membrane; a third enzyme responsible for transferring glucose to the secreted protein, collagen, is strongly bound to the plasma membrane (9). Like the HeLa cell studies (9), the present work has shown that the N-acetylgalactosaminyl and galactosyl transferases are tightly bound to the membranes of submaxillary glands. Unlike the HeLa cells, however, the two glycosyl transferases from submaxillary glands do not reside in the smooth internal membranes. It is possible that submaxillary gland glycosyl transferases are located in the plasma membrane, by analogy to the HeLa cell collagen: glucosyl transferase, because of their role in synthesis of secreted glycoproteins. The HeLa cell and submaxillary gland membranes were chosen for study, therefore, as representative of two different membranes containing the same glycosyl transferases. In other reports (ll-14), treatment with the nonionic detergent Triton X-100 was used to solubilize and purify the galactosyl and N-acetylgalactosaminyl transferases from their subcellular membrane sites in reticulocytes (13) and tumor cells (12). The main objective of this study was to investigate the effect of agents, particularly detergents, which lead to solubilization of the transferases by disruption of the membranes where they reside. Iciszer and De Robertis (10) were able to release acetylcholinesterase found in membrane material from the central nervous system by use of the nonionic detergent Triton X-100. In our study several nonionic and ionic agents were used to solubilize the transferases. In general, the nonionic detergents, representative of the polyoxyethylene series, solubilize the enzymes from the membranes as measured by the increase of activity over the UC treated membranes. MATERIALS
AND
METHODS
HeLa cell fractionation. The growth conditions for the HeLa cells and the method of cell rupture and fractionation are described in a previous report (15). In brief, the harvested HeLa cells were
AND
EYLAR
washed three times with 10-Z M calcium acetate in 0.15 M NaCl and were resuspended in 2 vol of 0.01 M EDTA in 0.02 Y Tris buffer, pH 7.2. The cells were then ruptured in a tight-fitting Dounce homogenizer with approximately 30 strokes. To remove whole cells, nuclei, mitochondria, and other contaminants, the homogenate was centrifuged at 4000 g for 10 min. The 4000 g supernatant fluid (4s) was fractionated in a discontinuous sucrose gradient. The 45 supernatant fluid was adjusted to 50% in sucrose and 20 ml were placed into a tube appropriate for the swinging bucket rotor 25.2 of the Spinco ultracentrifuge L-2. Thirteen milliliters of each of 40%, 35$!$,, and 30% sucrose solutions in 0.05 M Tris buffer, pH 7.2, were respectively layered over the sample. Finally 2 ml of 0.05 M Tris buffer, pH 7.2, was added. The gradient was centrifuged at 75,000 g (23,500 rpm) for 16 hr. Turbidity indicating membranous material was observed; each turbid layer was separately removed with pipettes. The six fractions were: S1 , 30yo sucrose layer; Sz , 3@3570 interface; SI , 35407, interface; S4 , 40-50yo interface; S5 , 50% sucrose layer; and Se , the pellet. All fractions were diluted 34-fold with 0.05 M Tris buffer, pH 7.2, and centrifuged at 70,000 g for 1 hr in order to remove sucrose and soluble proteins. The washed membrane pellets were resuspended in 0.05 M Tris buffer, pH 7.2, and assayed for various glycosyl transferases. Preparation of membranous material from submaxillary glands. The submaxillary glands were obtained from cows within 10 min after killing at the local slaughter house. The fractionation method for the isolation and characterization of the polypeptidyl:N-acetylgalactosaminyl transferase from bovine submaxillary glands was described previously (14). For the purposes of this work, the submaxillary glands were extracted overnight in 0.05 M KC1 at 4”. The homogenate was passed through cheesecloth and the filtrate was centrifuged at 10,000 g for 20 min. The pellet (1OP) which contained nuclei, mitochondria, membranous material, and cytoplasmic components, was fractionated in a discontinuous sucrose gradient similar to the one used for HeLa cells, except for lower percentages of sucrose. The pellet (1OP) was adjusted to 457, in sucrose; the other layers were 357,,, 307,, and 25yc in sucrose. After overnight centrifugation, 6 fractions of membranous material were removed separately, diluted 3-fold with 0.05 M Tris buffer, pH 7.2, and centrifuged at 70,000 g for 1 hr. The pellets from each fraction were assayed for the glycosyl transferases. The 6 fractions are referred to as follows: SI , obtained from 257, sucrose layer; S. , the band at 25307; interface; Sa , the band at 3Cr35LA interface; S4 , the band at 3&45”j, interface; S;, , the material in 45% sucrose;
GLYCOPROTEIN ss , the pellet. Fraction
Sd was fixed for electron microscopy as described earlier (15). Detergents. The detergents used in this study were obtained from various sources: the Triton series (X-100, W-30 and 770) from Rohn and Haas Co.; the Tergitols (15-S-9 and NPX) from Union Carbide; the Tweens (20, 40, 60, and 80) and the Brijs
(35 and 58) from Atlas
mington,
Delaware;
Chemical
the Nonidet
Inc.,
Wil-
Pa from Shell
Chemicals, U. K. Ltd.; the Lubrols (WX and MOA) from General Biochemicals; the Antarox BL-225 and BL-240 from General Aniline and Film Corp., New York; and the ionic detergents, sodium dodecyl sulfate (SDS) and deoxycholate (DOC) from Sigma Chemical Co. Snake venom from Trimeresuru,s $avorviridis was purchased from Sigma Chemical Co. Extraction of the membranes with various detergents. In order to extract the glycosyl transferases from the membranous materials isolated from sucrose gradients, 1% solutions of the detergents in 0.1 M Tris buffer, pH 7.2, were added in O.l-ml quantities to 0.5 ml (1 mg protein/ml) of the membrane preparations. As a control, the membrane preparations were extracted with this buffer alone. The mixtures were shaken at 4” for 1 hr. In order to determine the degree of solubilization, the extracts were centrifuged at 100,000 g for 1 hr. The supernatant solution was used for testing for glycosyl transferase activity. Our criteria of solubility is the failure of the enzymes to sediment when centrifuged for 1 hr at 100,000 g. An enzyme, snake venom phospholipase A, was also used to extract the glycosyl transferases. The venom was boiled for 5 min; the supernatant fluid from 5 mg of original venom containing the phospholipase A was added to 0.5 ml of the membrane preparation. After 1 hr, the mixture was centrifuged at 100,000 g and the supernatant fluid was assayed for the transferases. Enzyme assays. The enzyme assay conditions and the components of the incubation mixtures were optimal for the polypeptidyl:N-acetyl galactosaminyl and the glycoprotein:galactosyl transferases as described previously (9). transThe polypeptidyl: N acetylgalactosaminyl ferase. The complete incubation mixture (final volume 0.15 ml) contained the following: 10 11 UDPiv-acetylgalactosamineJ4C (1.25 mCi/mmole, .04 rmoles approximately 1 X 105 cpm), 10 ~1 of receptor (0.2 mg, prepared from BSM by removal of sialic acid and of N-acetylhexosamine), 20 ~1 of 0.25 M MnC12, 50 ~1 of 0.1 M Tris buffer, pH 7.2, 10 ~1 of 1.0% detergent (Triton X-100), and 50 ~1 of membrane preparations (enzyme). In order to assay for the solubilization of the enzymes by different, detergents, instead of the last three components of the incubation mixture (Tris buffer,
BIOSYSTHESIS detergent,
449
and enzyme), 100 ~1 of the 100,000 g
supernatant fluid-detergent mixtures were used. After incubation at 37” for 45 min, the reaction was terminated by the addition of 2.0 ml of 1% phosphotungstic acid in 0.5 N HCI and the proteinbound radioactivity determined as described previously (18). The glycoprotein:galactosyl transferase. The assay conditions were as follows: the complete incubation mixture (final volume 0.155 ml) contained the following: 10 ~1 of UDP-galactose-‘*C, (5 X lo4 cpm 0.12 pmoles), 20 11 of 0.25 M MnClz , 15 ~1 of al-acid glycoprotein receptor (0.75 mg, prepared by removal of sialic acid and galactose), 50 ~1 of 0.1 M Tris buffer, pH 7.2, 10 ~1 of 1.0% detergent (Triton X-100), and 50 pl of membrane preparations. When the membrane preparations were extracted with various detergents before the assay, 100 ~1 of the membrane-detergent mixture were used instead of the last three components. After incubation at 25” for 20 min, the reaction was terminated and the protein-bound radioactivity determined as above. RESULTS
The distribution
of two glycosyl transferuses from HeLa cells and bovine submaxillary glands. The total
in the membrane fractions
activities of the polypeptidyl: N-acetylgalactosaminyl and glycoprotein: galactosyl transferases found in the membrane fractions derived from HeLa cells and bovine
submaxillary glands (obtained from sucrose gradients) are shown in Fig. 1. The HeLa cell membrane fractions were derived from the supernatant fluid obtained after centrifugation
of the
cellular
homogenate
40008 as described previously
at
(9). From
.55-70 % of the total glycosyl t’ransferase acbivities of the homogenate are found in the supernatant fluid. Once the membrane fract,ions are obt’ained from the sucrose gradient,
they are centrifuged
at 70,OOOg for 1 hr 0.05 M Tris buffer, pH 7.2) in order to remove the sucrose and (after
l-4
dilution
with
adsorbed protein. ,4s shown in Fig. 1, both glycosyl transferases of HeLa cells are found predominantly in fractions $31 (65%) and
Sz (30 %) ; over 90 % of the activity
applied
to the sucrose gradient was recovered in these two fractions. Very little of the transferase activities were found in fraction Ss and essentially no activity was found in fractions Sa , Sj, and SE . On the other hand,
450
HAGOPIAN
--ANU
the glycosyl transferases are predominantly bound to the Sh fraction from the submaxillary glands; fractions S1, SZ , and So show very little activity. The membrane fractions & of HeLa cells and S+ of submaxillary
1 HsL0 cells
ml-h-
Bovine Submoxilkwy
Glands
Fraction
Number
FIG. 1. Total 14Ccpm transferred by two glyco-
protein:glycosyl transferases located in various membrane fractions of HeLa cells (top) and bovine submaxillary glands (bottom). The first (left) vertical bar of each set of two refers to 1% cpm transferred by polypeptidyl:galNAc and the second bar (on right) refers to glycoprotein:galactosyl transferases.
glands also show the highest specific activities (cpm/mg protein) of the transferases (Table I). It is apparent that preparation of the S1 and Sbfractions of HeLa cell and submaxillary membranes lead to at least a 20-50-fold and S-lo-fold purification, respectively, of the glycosyl transferases over the original homogenates. As described previously, electron micrographs of HeLa fraction S1 show only smooth internal membranes (15) ; electron micrographs of submaxillary gland Sq fraction showed no nuclear contamination, but some mitochondrial fragments and rough membranes were observed. It was noted that glycosyl transferases shown in Fig. 1 and Table I were firmly bound to membranes (& and Sh) ; the enzymes sedimented with the membranes at 70,OOOg. Since most of the glycosyl transferase activities are bound to fractions S1 of HeLa cells and S4 of submaxillary glands which contain also the highest specific activities, these two fractions were used to study the solubilization of the enzymes produced by various detergents. Detergent extraction of the glycosyl transferases from membrane fractions SI of HeLa cells and 84 of submaxillary glands. The results of solubilization of the glycosyl transferases by various detergents and phospholipase A from membrane fractions of S1 (HeLa) and 54 (bovine submaxillary glands) are presented in Table II. In order to evaluate the relative influence of the different
TABLE THE SPECIFIC ACTIVITIES VARIOUS MEMBRANE
(CPM/MG
FRACTIONS
---EYLAK
I
OF Two GLYCOSYL TRANSFERASES LOCATED IN FROM HELA CELLS AND BOVINE SUBMAXILLARY GLANDS PROTEIN)
Bovine submaxillary glands
HeLa cells Fraction
Polypeptidyl: galNAc transferase
Supernatant fluid (45) Pellet (1OP) Sl S* S, S4 SS SS
Glycoprothxgal transferase
500
200
9600 2500 2300 2000 700 0
9600 2400 2300 1000 200 0
Polypeptidyl: galNAc transferase
4000 3000 3000 3000 34,000 10,000 3000
Glycoprotekgal transferase
3000 2000 2000 3000 34,000 3000 3000
GLYCOPROTEIN
detergents, the 100,OOOgsupernatant fluids of the membrane fractions extracted with buffer alone or with detergents were assayed for t,he glycosyl transferases. The enzyme activities are given as cpm/ml of supernatant fluid. The increase in activity due t’o the presence of detergents is shown by the ratio of activities in the presence and absence of detergents. Most of the nonionic detergents test’ed were effective in extracting t’he glycosyl t’ransferases. In the extraction of N-acetylgalactosaminyl transferase from HeLa fraction the most effective detergents were Sl T&on X-100 and Tergitol NPX. Compared to Triton X-100, Brij 58, Lubrol WX, and Nonidet PqO were 85 %, 72%, and 70% as effective, respect’ively, while Tergitol 15-S-9 and Tweens 20 and 40 were approximately
40-50 % as effective. Tweens 60 and 80 and Lubrol 1\IOA gave results similar to that obtained with Tris buffer. The enzyme was totally inactivated by the Tritons W30 and 770 and the ionic detergents deoxycholate and sodium dodecyl sulfate. Phospholipase A extracted 52% as much of this enzyme as did the best detergent, Triton X-100. In the case of the galactosyl transferase extraction from HeLa membranes S1, the Nonidet PbO detergent was the most effective. Compared to Nonidet Phg , Brij 55 and Lubrol WX were 77 % as effective; Triton X-100, Tergitols NPX and 15-S-9 were approximately 62-66%; Tween 20, 44 %; Tween 40, 30%, and Tween 60 and Lubrol MOA, 20% as effective. Phospholipase A was 26 % as effective as the best detergent. From the S1
TABLE
II
THE EFFECT OF DETERGENTS ON THE ACTIVITY TRANSFERASES OF THE HELA CELL MEMBRANE GLAND MEMBR:\NE
OF Two GLYCOPROTEIN : GLYCOSYL S, AND BOVINE SUBMAXILLARY
Sa
Transferases in HeLa S1 membrane Detergent?’
Polypeptidyl: galNAc transfera% Cpm/ml X 10-z
None (0.1 M Tris, pII 7.2) Triton X-100 Tergitol NPX Tergitol 15-S-9 Brij 58 Brij 35 Nomdet Pdg Lubrol WX Lubrol MOA Antarox BL-225 Antarox BL-240 Tween 20 Tween 40 Tween 60 Tween 80 Deoxycholate Phospholipase A Cetylpyridinium Bromide
2.94 12.8 12.5 5.1 10.9 -6 8.9 9.3 2.1 5.8 6.0 2.3 3.4 0.0 6.7 0.3
Ratiob
Transferases in submaxillary gland Sd membrane
Glycoprotein:gal transferase CPw$
451
BIOSYNTHESIS
X
Ratioh
Polypeptidyl:gaWAc transfera%? CPm$J
X
Glycoprotekgal transferase
Ratiob
“P;$F/
X
R&oh
1.0
3.9
1.0
3.7
1.0
5.8
1.0
4.4 4.3 1.7 3.7
45.4 48.2 44.8 56.5
11.6 12.3 11.5 14.4 -
3.1 3.2 0.7 -
73.3 56.5 14.9 -
18.7 14.4 3.8 -
2.0 2.1 0.8 1.2 0.0 2.3 0.1
32.5 20.7 14.7 19.2 1.0
34.3 36.9 27.8 29.1 20.0 33.0 29.6 4.9 26.1 15.6 12.0 13.4 10.6 14.3 9.8 8.3 1.1
9.3 10.0 7.5 7.9 5.4 8.9 9.0 1.3 7.0 4.2 3.3 3.6 2.9 3.9 2.6 2.2 0.3
42.4 45.6 49.5 38.3 27.7 46.5 49.6 5.7 41.1 41.6 25.0 12.6 9.8 14.0 2.2 11.6 2.9
7.3 7.9 8.5 6.6 4.8 8.0 8.5 1.0 7.1 7.2 4.3 2.2 1.7 2.4 0.4 2.0 0.5
8.3 5.6 3.8 5.0 0.25
0 Detergents Tritons W30 and 770 and sodium dodecyl sulfate tivities are close to 0. b Ratio refers to the activity (+ detergent/ - detergent). c These marks refer to activities not measured.
are not listed
becanse the resulting
ac-
452
HAGOPIAN
membrane preparation of HeLa cells, the maximum increase in specific activity was about four times greater for the glycoprotein: galactosyl than the polypeptidyl: Nacetylgalactosaminyl transferase. Bovine submaxillary membrane preparations (4S), when extracted with detergents, released both glycosyl transferases; the maximum increase in specific activity in this case was approximately S-lo-fold and compares with the HeLa cell preparations where a 4-l&fold increase was observed. As in HeLa cells, the detergents most effective for the release of galNAc transferase were Tergitol NPX and Triton X-100. The detergents Nonidet P40 , Lubrol WX, Brij 58, Tergitol 15-S-9, and Antarox BT,225 were 70-90 % as effective as the Tergitol NPX. The Tween series, Antarox BL-240, and Brij 35 solubilized approximat’ely 3054% as much enzyme activity as the best, detergent. Lubrol MOA and Triton 770 produced no effect over the Tris buffer while Triton W30 and sodium dodecyl sulfate totally inactivated the transferase. Phospholipase A and the ionic detergent deoxycholate had about 25 % the effect of Tergitol NPX. The best detergents for the release of the galactosyl transferase from St submaxillary membranes were Tergitol 15-S-9 and Lubrol WX. Also very effective were Nonidet P4,, (94% of Tergitol 15-S-9), Tergitol NPX (93 %), Triton X-100 (86 %), Ant,arox BL-240 (85 %), and BL-225 (84 %). Tritons W30 and 770 and SDS inactivated the galactose transferase while all other detergents used either had no effect or were less than 50% as effective as the best detergent.
The e$ect of the detergents on the solubilixed and puri$ed N-acetylgalactosaminyl transferase. In a preliminary step in the purification process
of the galNAc
transferase,
the
bovine submaxillary glands are extracted with Triton X-100 (14). This process releases the enzyme in soluble form and further purification is achieved by differential centrifugation and gel filtration (80-fold). To test the effect of the various detergents on the purified enzyme, the Triton X-100 already present was removed by gel filtration by Sephadex G-100 column chromatog-
AND EYLAR
III 0.8 2 0.6 ?i 6
0.4 l$
5
10
20
15 Tubs
25
30
I
0.2
Number
FIG. 2. Separation of purified polypeptidyl: galNAc from Triton X-100 by Sephadex G-100 column chromatography. The fractions were eluted with 0.05 M Tris buffer, pH 7.2. Each fraction was assayed for polypeptidyl:galNAc transferase activity (0-O). The optical density at 280 rnp of each fraction was also measured ( l --0). TABLE
III
THE EFFECT OF THE DETERGENTS ON THE TOTAL ACTIVITY OF THE PURIIVED POLYPEPTIDYL: galNAc TRANSFERASE ISOLATED FROM BOVINE SUBMAXILLARY GLANDS Detergent
Cpm/ml x 10-a
None (0.1 M Tris, pH 7.2) Triton X-100 Tween 20 Tween 40 Tween 60 Tween 80 Nonidet PRO Brij 58 Tergitol 15-S-9 Tergitol NPX Lubrol MOA Lubrol WX Triton 770 Triton W30 Sodium dodecyl sulfate Deoxycholate
14.7 20.4 14.0 15.7 11.2 12.3 10.1 14.8 10.6 12.2 14.6 13.0 3.0 0 0 0
raphy. Figure 2 shows the separation of the enzyme from the Triton X-100 detergent. The tubes containing the transferase were combined, lyophilized, and dialyzed against .005 M Tris buffer, pH 7.2. Table III shows the effect, of the various detergents on the purified polypeptidyl: galNAc transferase.
GLYCOPIIOTEIN
All the nonionic detergents had no deletcrious or advantageous effect on the enzyme activity when compared to the control, in which case no detergent, but Tris buffer, was added. On the other hand, the ionic detergent,s sodium dodecgl sulfate, sodium deoxpcholate, Trit,on W30, and Triton $70 tot,ally inactivated t’he galP\‘Ac transferase.
HIOSYNTHEYIS
453
bmnes of the HeLa cells (&) and the membrane fraction (S,) of the submaxillary tissue is demonstrated by both the yield and degree of purificat,ion (Table I). The main finding of t,his study is that regardless of the type of membrane in which t,he glgcosyl transferases are found, they appear to be intimately bound within the membranous framework; disruption of the DISCUSSION membranous structure is necessary for full expression of activity. From SO-95% of The object of this study was to investigat,e the total enzyme activities are masked in the dissociation of glycosyl transferases from their membrane environment where they are the isolat,ed membranes compared to the opintimat,ely bound. E’or this purpose, two timal conditions produced by the nonionic glycoprotein: glycosyl t)ransferases appeared detergents. This finding suggests that the in situ localization of the transferases as appropriate: the polypeptidyl:galNAc transferase, which is responsible for synthesis of part of the membrane structure represents an important aspect of their in vivo functhe protein-carbohydrate linkage involving tion. The data also suggest that nonpolar hydroxyamino acids linked glycosidically binding plays an important role in the to N-acetylgalactosamine (l-2), and the gslactosyl transferase which is specific for interaction of the transferases with their membranous environment. It is of interest synthesis of the /3-n-galactosyl-(M-Nin this regard that in both types of memucetylglucosamine linkage in glycoproteins branes (fraction S1 from HeLa or fraction (I&19). Although these two enzymes show a Sb from submaxillary glands) the transhigh specificity for their receptors, the resultant’ glgcoprotein product may vary de- ferases appear to respond similarly to the pending on the choice of tissue. For example, various detergents. The nonionic detergents Triton X-100, Tergitol NPX and 15-S-9, in the HeLa cells these enzymes are involved Nomdet Pqg , and Lubrol WX are most efin the assembly of membrane glycoproteins, fect,ive in general and lead to maximal acwhereas in t,he submaxillary gland they part,icipate in the synt’hesis of secreted tivity. In general, the detergents show the same relative effect on each enzyme from submaxillary glycoprot’eins. For comparathe two types of membranes; the Tween tive purposes, therefore, HeLa cell transferases were chosen for comparison with the series lead to intermediate activities and results with sodium analogous submaxillary transferases. As seen complete inhibition dodecyl sulfate and Triton W30, which are from Table I, the cellular localization of negatively charged molecules. t.hese enzymes differ depending on the With respect t,o the action of the various choice of tissue; in HeLa cells, they are found in the smooth internal membranes as detergents, it is of interest to compare detergent structure with the activities found part of a multienzyme group of transferases for the transferases. The nonionic deter(9), whereas in the submaxillary tissue they gents are of t’he polyoxyethylene type. The are found near the 40-50% sucrose intermost effective det,ergents, such as Triton face. It is possible in the latter case that X-100, Tergitol NPX, and Nonidet PbOare the glycosyl t’ransferases are located in the of the polyoxyethylene octylphenol type; plasma membranes which migrate to t.his slightly less effective are the linear primary position during the isopyenie centrifugaalcohol polyoxyethylene types such as tion. Such is the case for the collagen: Antarox BL-225 and Brij 35. The Tween glucosyl transferase found in the HeLa cells (9), which is involved in synthesis of detergents, which are generally less than collagen, a secreted glycoprotein. It is 50% as effective as the octylphenol series, noteworthy that the localization of the are polyoxyethylene sorbitan monofatty glycosyl t,ransferases in the smooth mem- acids. Thus, the grouping to which the
polyoxyethylene chain is attached has considerable influence on the ability of the detergent to disrupt nonpolar membrane structures and solubilize the glycosyl transferases. Those detergents containing the alkylphenol grouping appear to be most effective. The influence of charged groups on the basic nonpolar detergent molecule are clearly illustrated by Triton W30 and Triton 770 which are sulfonated or sulfated polyoxyethylene alkylphenols, respectively. Both result in complete inhibition in nearly every case. The other negatively charged molecules, such as sodium dodecyl sulfate and deoxycholate, also give a high degree of inhibition. The inhibitory action of charged detergents is not restricted to the negatively charged molecules, but also is found with cetylpyridinium bromide, a detergent with a positive charge. In addition to detergents, membrane dissociation with phospholipase A also resulted in partial release of the transferases. With the N-acetylgalactosaminyl transferase from HeLa cells, the phospholipase A treatment increased the activity nearly 5-fold. In view of the action of the phospholipase A, which leads to release of fatty acid from the phospholipids of the membrane, it appears that nonpolar forces play a significant role in membrane substructure. This result is comparable with the recent subunit concept of membrane structure in contrast to the Davson-Danielli unit membrane hypothesis (16) ; a similar conclusion was derived by Lenard and Singer (17) using erythrocyte membranes and phospholipase C. In evaluation of the initial influence of detergents on the transferases, the resultant activity appears to depend on two factors, solubilization and inhibition. In order to answer questions concerning the activation or inhibition of detergents on the glycosyl transferases as well as the possible nonpolar stabilization of these enzymes once separated from their membranous environment, polypeptidyl: N-acetylgalactosaminyl the transferase was subjected to gel filtration in order to remove the detergent. Using the polypeptidyl: N-acetylgalactosaminyl transferase purified from submaxillary gland membranes it is evident that the enzyme
does not require the presence of detergent for full activity. Upon removal of the Triton X-100, the enzyme activities were approximately the same with or without detergent. Although the adsorption of a low level of Triton X-100 cannot be discounted, it appears that most of the detergents neither inhibit nor activate this enzyme. It can be concluded, for example, that the failure of Tween 80 to give full activity is due to inefficient solubilization. It is apparent, however, that the complete inactivity in the presence of negatively charged detergents, such as Triton W30, sodium dodecyl sulfate, and sodium deoxycholate, is due to inactivation of the enzyme rather than a lack of solubilization. It is of interest that these polar detergents, which are commonly used in biochemical studies, lead to inactivation, whereas the nonpolar detergents are without influence generally. Furthermore, the enzyme prepared in this manner by gel filtration is stable to lyophilization and freezing even over a period of several months and, thus, does not require detergent stabilization. It appears, therefore, that the main effect of the nonpolar detergents on the membranes with respect to the glycosyl transferases is release and solubilization. The detergents are not required for activation or stabilization. It cannot be decided from this study if the different effects produced by the ionic and nonionic detergents is due to a difference in micellar structure, direct inactivation of the enzymes, or to some other factor. The transferases of this report contrast with those found by Roseman and co-workers which are not membrane bound, but exist normally in a soluble form in goat colostrum (E-20). In a preliminary study, Schachter and McGuire (21) reported on a glycosyl transferase from porcine submaxillary gland that is particulate bound. ACKNOWLEDGMENTS We are indebted to Mr. Jesse Jackson and Mr. Roger Birosel for valuable technical assistance and to Mr. Donald Wegemer and Mrs. Chris Law for growing the HeLa cells used in this work. We also thank Dr. Jonas Salk for helpful advice and encouragement.
GLYCOPROTEIN
BIOSYNTHESIS
REFERENCES 1. SARCIONE, E. (1964).
J., J.
Biol.
Chem. 239, 1686
2. MOLNAR, J., ROBINSON, G. B., AND WINZLER, R. J., J. Biol. Chem. 240,1882 (1962).
3. O’BRIEN, P. J., CANADY, M. R., HALL, C. W., AND NEUFELD, E. F., Bioehem. Biophys. Acta 117, 331 (1966). 4. SIMBIN, J. L., AND JAMIESON, J. C., Biochem. J. 103, 38P (1967). 5. LA~FORD, G. R., AND SCHACHTER, H., J. Biol. Chem. 241, 5408 (1966). 6. LI, Y., LI, S., AND SHELTER, M. R., J. Biol. Chem. 243, 656 (1968). 7. COOK, G. M. W., LAICO, M. T., AND EYLAR, E. H., Proc. Natl. Acad. Sci. U. S. 64, 247 (1965). 8. BOUCHILLOUX, S., AND CHEFTEL, C., Biochem. Biophys. Res. Commun. 23,307 (1966). 9. HAGOPIAN, A., BOSMANN, H. B., AND EYLAR, E. H., Arch. Biochem. Biophys. 128,387 (1968). 10. FISZER, S., AND DEROBERTIS, E., Brain Res. 6, 31 (1967). 11. EYLAR, E. H., CACUM, J. F., HAGOPIAN, A.,
12. 13. 14. 15.
16. 17. 18.
19.
20.
21.
455
AND LAICO, M. T., Abstr. IV Intern. Congr. Biochem. (1967). CACCAM, J. F., AND EYLAR, E. H., in preparation. LAICO, M. T., AND EYLAR, E. H., Federation Proc. 26, 2252 (1966). HAGOPIAN, A. AND EYLAR, E. H., Arch. Biothem. Biophys. 129,515 (1969). BOSMANN, H. B., HAGOPIAN, A., AND EYLAR, E. H., Arch. Biochem. Biophys. (1968). In press. KORN, E. D., Science 163,149l (1966). LENARD, J., AND SINGER, S. J., Science 169, 738 (1968). BARTHOLOMEW, B., JOURDIAN, G. W., AND ROSEMAN, S., Abstr. 6th Intern. Congr. Biothem. IUB 32,503, vi-i (1964). MCGUIRE, E. J., JOURDIAN, G. W., CARLSON, D. M., AND ROSEMAN, S., 1. Biol. Chem. 240, PC 4112 (1965). JOHNSTON, I. R., MCGUIRE, E. J., JOURDIAN, G. W., AND ROSEMAN, S., J. Biol. Chem. 241, 5735 (1966). SCHACHTER, H., AND MCGUIRE, E. J., Pederation Proc. 27, 345 (1968).
OF
BIOCHEMISTRY
Glycoprotein
AND
BIOPHYSICS
Biosynthesis: from
447-455 (1969)
12%
The Solubilization
Membranes
of Hela
Submaxillary ARPI
Received
June
Cells
and
Glycoproteins”
HAGOPIAN
The Salk Institute
of Glycosyl
AND
for Biological
Bovine 2
E. H. EYLAR
Studies,
10, 1968; accepted
Transferases
San Diego, October
California
21, 1968
Two glycosyl transferases, the polypeptidyl:N-acetylgalactosaminyl and glycoprotein:galactosyl transferase, which participate in the assembly of the carbohydrate units of glycoproteins, were purified from both HeLa cells and submaxillary glands. In the case of HeLa cells these enzymes are intimately associated with the smooth internal membranes where they form part of the multienzyme group of transferases synthesizing membrane glycoproteins; in submaxillary tissues, however, these enzymes are involved in synthesis of secreted submaxillary glycoproteins and are strongly bound to membranes, possibly plasma membranes, which sediment, to the 3&45yo sucrose interface. In preparing the respective membranes, the glycosyl transferases were purified from l&40-fold over the cellular homogenate. It was found that from SO-95% of the total enzyme activities are masked in the isolated membranes; disruption of the membranous structure with nonionic detergents or with phospholipase A liberated the transferases. The largest increase in activity was obtained with the polyoxyethylene octylphenol type. The linear alcohol polyoxyethylene types were slightly less effective; less than 50% of the maximum activity was obtained. These results, together with the effect of phospholipase A, emphasize the significance of nonpolar forces in membrane substructure, particularly with regard to the interaction of the transferases with their membranous environment. By contrast, negatively charged detergents, such as sodium deoxycholate and sodium dodecyl sulfate, led to strong inhibition of enzymes. A positively charged detergent, cetylpyridinium bromide, also produced strong inhibition. It was concluded that the influence of detergents on the activity of membrane-bound transferases appears to depend on two factors, solubilization and inhibition.
In Dhe biosynthesis of glycoproteins, t’he steps leading to synthesis of polypeptide chain and formation of t’he carbohydrate 1 This study was supported, in part, by a grant from The National Foundation to Dr. Jonas Salk, and in part, by the American Cancer Society, California Division. 2 The terminology used in this report in referring to individual glycosyl transferases designates the macromolecular receptor and the monosaccharide being transferred, e.g., polypeptidyl:X-acetylgalactosaminyl transferase. The following abbreviations were used: galNAc, Nacetylgalactosamine; gal, galactose; SDS, sodium dodecyl sulfate; DOC, sodium deoxycholate; BSM, bovine submaxillary glycoprotein; UDP, uridine diphosphate. 447
units and the integration of these two processes must be considered. Present data (l-7) suggest that the synthesis of the polypeptide is carried out at the polyribosomes according to current concepts for simple proteins. Controversy still exists over the role of microsomes or ribosomes as a site for attachment of the carbohydrate moiety to the protein (5, 6,s). A multienzyme group of transferases involved in the biosynthesis of carbohydrate units of membrane glycoproteins has been found (9). It is reasonable to assume that the cellular location of the transfcrases dictates the site of attachment of monosaccharides to the polypeptide chain. In an earlier study with HeLa cells
44s
HAGOPIAN
it was shown that two enzymes involved in biosynthesis of membrane glycoproteins, the polypeptidyl: N-acetylgalactosaminyl and glycoprotein: galactosyl transferases are located in the same internal smooth membrane; a third enzyme responsible for transferring glucose to the secreted protein, collagen, is strongly bound to the plasma membrane (9). Like the HeLa cell studies (9), the present work has shown that the N-acetylgalactosaminyl and galactosyl transferases are tightly bound to the membranes of submaxillary glands. Unlike the HeLa cells, however, the two glycosyl transferases from submaxillary glands do not reside in the smooth internal membranes. It is possible that submaxillary gland glycosyl transferases are located in the plasma membrane, by analogy to the HeLa cell collagen: glucosyl transferase, because of their role in synthesis of secreted glycoproteins. The HeLa cell and submaxillary gland membranes were chosen for study, therefore, as representative of two different membranes containing the same glycosyl transferases. In other reports (ll-14), treatment with the nonionic detergent Triton X-100 was used to solubilize and purify the galactosyl and N-acetylgalactosaminyl transferases from their subcellular membrane sites in reticulocytes (13) and tumor cells (12). The main objective of this study was to investigate the effect of agents, particularly detergents, which lead to solubilization of the transferases by disruption of the membranes where they reside. Iciszer and De Robertis (10) were able to release acetylcholinesterase found in membrane material from the central nervous system by use of the nonionic detergent Triton X-100. In our study several nonionic and ionic agents were used to solubilize the transferases. In general, the nonionic detergents, representative of the polyoxyethylene series, solubilize the enzymes from the membranes as measured by the increase of activity over the UC treated membranes. MATERIALS
AND
METHODS
HeLa cell fractionation. The growth conditions for the HeLa cells and the method of cell rupture and fractionation are described in a previous report (15). In brief, the harvested HeLa cells were
AND
EYLAR
washed three times with 10-Z M calcium acetate in 0.15 M NaCl and were resuspended in 2 vol of 0.01 M EDTA in 0.02 Y Tris buffer, pH 7.2. The cells were then ruptured in a tight-fitting Dounce homogenizer with approximately 30 strokes. To remove whole cells, nuclei, mitochondria, and other contaminants, the homogenate was centrifuged at 4000 g for 10 min. The 4000 g supernatant fluid (4s) was fractionated in a discontinuous sucrose gradient. The 45 supernatant fluid was adjusted to 50% in sucrose and 20 ml were placed into a tube appropriate for the swinging bucket rotor 25.2 of the Spinco ultracentrifuge L-2. Thirteen milliliters of each of 40%, 35$!$,, and 30% sucrose solutions in 0.05 M Tris buffer, pH 7.2, were respectively layered over the sample. Finally 2 ml of 0.05 M Tris buffer, pH 7.2, was added. The gradient was centrifuged at 75,000 g (23,500 rpm) for 16 hr. Turbidity indicating membranous material was observed; each turbid layer was separately removed with pipettes. The six fractions were: S1 , 30yo sucrose layer; Sz , 3@3570 interface; SI , 35407, interface; S4 , 40-50yo interface; S5 , 50% sucrose layer; and Se , the pellet. All fractions were diluted 34-fold with 0.05 M Tris buffer, pH 7.2, and centrifuged at 70,000 g for 1 hr in order to remove sucrose and soluble proteins. The washed membrane pellets were resuspended in 0.05 M Tris buffer, pH 7.2, and assayed for various glycosyl transferases. Preparation of membranous material from submaxillary glands. The submaxillary glands were obtained from cows within 10 min after killing at the local slaughter house. The fractionation method for the isolation and characterization of the polypeptidyl:N-acetylgalactosaminyl transferase from bovine submaxillary glands was described previously (14). For the purposes of this work, the submaxillary glands were extracted overnight in 0.05 M KC1 at 4”. The homogenate was passed through cheesecloth and the filtrate was centrifuged at 10,000 g for 20 min. The pellet (1OP) which contained nuclei, mitochondria, membranous material, and cytoplasmic components, was fractionated in a discontinuous sucrose gradient similar to the one used for HeLa cells, except for lower percentages of sucrose. The pellet (1OP) was adjusted to 457, in sucrose; the other layers were 357,,, 307,, and 25yc in sucrose. After overnight centrifugation, 6 fractions of membranous material were removed separately, diluted 3-fold with 0.05 M Tris buffer, pH 7.2, and centrifuged at 70,000 g for 1 hr. The pellets from each fraction were assayed for the glycosyl transferases. The 6 fractions are referred to as follows: SI , obtained from 257, sucrose layer; S. , the band at 25307; interface; Sa , the band at 3Cr35LA interface; S4 , the band at 3&45”j, interface; S;, , the material in 45% sucrose;
GLYCOPROTEIN ss , the pellet. Fraction
Sd was fixed for electron microscopy as described earlier (15). Detergents. The detergents used in this study were obtained from various sources: the Triton series (X-100, W-30 and 770) from Rohn and Haas Co.; the Tergitols (15-S-9 and NPX) from Union Carbide; the Tweens (20, 40, 60, and 80) and the Brijs
(35 and 58) from Atlas
mington,
Delaware;
Chemical
the Nonidet
Inc.,
Wil-
Pa from Shell
Chemicals, U. K. Ltd.; the Lubrols (WX and MOA) from General Biochemicals; the Antarox BL-225 and BL-240 from General Aniline and Film Corp., New York; and the ionic detergents, sodium dodecyl sulfate (SDS) and deoxycholate (DOC) from Sigma Chemical Co. Snake venom from Trimeresuru,s $avorviridis was purchased from Sigma Chemical Co. Extraction of the membranes with various detergents. In order to extract the glycosyl transferases from the membranous materials isolated from sucrose gradients, 1% solutions of the detergents in 0.1 M Tris buffer, pH 7.2, were added in O.l-ml quantities to 0.5 ml (1 mg protein/ml) of the membrane preparations. As a control, the membrane preparations were extracted with this buffer alone. The mixtures were shaken at 4” for 1 hr. In order to determine the degree of solubilization, the extracts were centrifuged at 100,000 g for 1 hr. The supernatant solution was used for testing for glycosyl transferase activity. Our criteria of solubility is the failure of the enzymes to sediment when centrifuged for 1 hr at 100,000 g. An enzyme, snake venom phospholipase A, was also used to extract the glycosyl transferases. The venom was boiled for 5 min; the supernatant fluid from 5 mg of original venom containing the phospholipase A was added to 0.5 ml of the membrane preparation. After 1 hr, the mixture was centrifuged at 100,000 g and the supernatant fluid was assayed for the transferases. Enzyme assays. The enzyme assay conditions and the components of the incubation mixtures were optimal for the polypeptidyl:N-acetyl galactosaminyl and the glycoprotein:galactosyl transferases as described previously (9). transThe polypeptidyl: N acetylgalactosaminyl ferase. The complete incubation mixture (final volume 0.15 ml) contained the following: 10 11 UDPiv-acetylgalactosamineJ4C (1.25 mCi/mmole, .04 rmoles approximately 1 X 105 cpm), 10 ~1 of receptor (0.2 mg, prepared from BSM by removal of sialic acid and of N-acetylhexosamine), 20 ~1 of 0.25 M MnC12, 50 ~1 of 0.1 M Tris buffer, pH 7.2, 10 ~1 of 1.0% detergent (Triton X-100), and 50 ~1 of membrane preparations (enzyme). In order to assay for the solubilization of the enzymes by different, detergents, instead of the last three components of the incubation mixture (Tris buffer,
BIOSYSTHESIS detergent,
449
and enzyme), 100 ~1 of the 100,000 g
supernatant fluid-detergent mixtures were used. After incubation at 37” for 45 min, the reaction was terminated by the addition of 2.0 ml of 1% phosphotungstic acid in 0.5 N HCI and the proteinbound radioactivity determined as described previously (18). The glycoprotein:galactosyl transferase. The assay conditions were as follows: the complete incubation mixture (final volume 0.155 ml) contained the following: 10 ~1 of UDP-galactose-‘*C, (5 X lo4 cpm 0.12 pmoles), 20 11 of 0.25 M MnClz , 15 ~1 of al-acid glycoprotein receptor (0.75 mg, prepared by removal of sialic acid and galactose), 50 ~1 of 0.1 M Tris buffer, pH 7.2, 10 ~1 of 1.0% detergent (Triton X-100), and 50 pl of membrane preparations. When the membrane preparations were extracted with various detergents before the assay, 100 ~1 of the membrane-detergent mixture were used instead of the last three components. After incubation at 25” for 20 min, the reaction was terminated and the protein-bound radioactivity determined as above. RESULTS
The distribution
of two glycosyl transferuses from HeLa cells and bovine submaxillary glands. The total
in the membrane fractions
activities of the polypeptidyl: N-acetylgalactosaminyl and glycoprotein: galactosyl transferases found in the membrane fractions derived from HeLa cells and bovine
submaxillary glands (obtained from sucrose gradients) are shown in Fig. 1. The HeLa cell membrane fractions were derived from the supernatant fluid obtained after centrifugation
of the
cellular
homogenate
40008 as described previously
at
(9). From
.55-70 % of the total glycosyl t’ransferase acbivities of the homogenate are found in the supernatant fluid. Once the membrane fract,ions are obt’ained from the sucrose gradient,
they are centrifuged
at 70,OOOg for 1 hr 0.05 M Tris buffer, pH 7.2) in order to remove the sucrose and (after
l-4
dilution
with
adsorbed protein. ,4s shown in Fig. 1, both glycosyl transferases of HeLa cells are found predominantly in fractions $31 (65%) and
Sz (30 %) ; over 90 % of the activity
applied
to the sucrose gradient was recovered in these two fractions. Very little of the transferase activities were found in fraction Ss and essentially no activity was found in fractions Sa , Sj, and SE . On the other hand,
450
HAGOPIAN
--ANU
the glycosyl transferases are predominantly bound to the Sh fraction from the submaxillary glands; fractions S1, SZ , and So show very little activity. The membrane fractions & of HeLa cells and S+ of submaxillary
1 HsL0 cells
ml-h-
Bovine Submoxilkwy
Glands
Fraction
Number
FIG. 1. Total 14Ccpm transferred by two glyco-
protein:glycosyl transferases located in various membrane fractions of HeLa cells (top) and bovine submaxillary glands (bottom). The first (left) vertical bar of each set of two refers to 1% cpm transferred by polypeptidyl:galNAc and the second bar (on right) refers to glycoprotein:galactosyl transferases.
glands also show the highest specific activities (cpm/mg protein) of the transferases (Table I). It is apparent that preparation of the S1 and Sbfractions of HeLa cell and submaxillary membranes lead to at least a 20-50-fold and S-lo-fold purification, respectively, of the glycosyl transferases over the original homogenates. As described previously, electron micrographs of HeLa fraction S1 show only smooth internal membranes (15) ; electron micrographs of submaxillary gland Sq fraction showed no nuclear contamination, but some mitochondrial fragments and rough membranes were observed. It was noted that glycosyl transferases shown in Fig. 1 and Table I were firmly bound to membranes (& and Sh) ; the enzymes sedimented with the membranes at 70,OOOg. Since most of the glycosyl transferase activities are bound to fractions S1 of HeLa cells and S4 of submaxillary glands which contain also the highest specific activities, these two fractions were used to study the solubilization of the enzymes produced by various detergents. Detergent extraction of the glycosyl transferases from membrane fractions SI of HeLa cells and 84 of submaxillary glands. The results of solubilization of the glycosyl transferases by various detergents and phospholipase A from membrane fractions of S1 (HeLa) and 54 (bovine submaxillary glands) are presented in Table II. In order to evaluate the relative influence of the different
TABLE THE SPECIFIC ACTIVITIES VARIOUS MEMBRANE
(CPM/MG
FRACTIONS
---EYLAK
I
OF Two GLYCOSYL TRANSFERASES LOCATED IN FROM HELA CELLS AND BOVINE SUBMAXILLARY GLANDS PROTEIN)
Bovine submaxillary glands
HeLa cells Fraction
Polypeptidyl: galNAc transferase
Supernatant fluid (45) Pellet (1OP) Sl S* S, S4 SS SS
Glycoprothxgal transferase
500
200
9600 2500 2300 2000 700 0
9600 2400 2300 1000 200 0
Polypeptidyl: galNAc transferase
4000 3000 3000 3000 34,000 10,000 3000
Glycoprotekgal transferase
3000 2000 2000 3000 34,000 3000 3000
GLYCOPROTEIN
detergents, the 100,OOOgsupernatant fluids of the membrane fractions extracted with buffer alone or with detergents were assayed for t,he glycosyl transferases. The enzyme activities are given as cpm/ml of supernatant fluid. The increase in activity due t’o the presence of detergents is shown by the ratio of activities in the presence and absence of detergents. Most of the nonionic detergents test’ed were effective in extracting t’he glycosyl t’ransferases. In the extraction of N-acetylgalactosaminyl transferase from HeLa fraction the most effective detergents were Sl T&on X-100 and Tergitol NPX. Compared to Triton X-100, Brij 58, Lubrol WX, and Nonidet PqO were 85 %, 72%, and 70% as effective, respect’ively, while Tergitol 15-S-9 and Tweens 20 and 40 were approximately
40-50 % as effective. Tweens 60 and 80 and Lubrol 1\IOA gave results similar to that obtained with Tris buffer. The enzyme was totally inactivated by the Tritons W30 and 770 and the ionic detergents deoxycholate and sodium dodecyl sulfate. Phospholipase A extracted 52% as much of this enzyme as did the best detergent, Triton X-100. In the case of the galactosyl transferase extraction from HeLa membranes S1, the Nonidet PbO detergent was the most effective. Compared to Nonidet Phg , Brij 55 and Lubrol WX were 77 % as effective; Triton X-100, Tergitols NPX and 15-S-9 were approximately 62-66%; Tween 20, 44 %; Tween 40, 30%, and Tween 60 and Lubrol MOA, 20% as effective. Phospholipase A was 26 % as effective as the best detergent. From the S1
TABLE
II
THE EFFECT OF DETERGENTS ON THE ACTIVITY TRANSFERASES OF THE HELA CELL MEMBRANE GLAND MEMBR:\NE
OF Two GLYCOPROTEIN : GLYCOSYL S, AND BOVINE SUBMAXILLARY
Sa
Transferases in HeLa S1 membrane Detergent?’
Polypeptidyl: galNAc transfera% Cpm/ml X 10-z
None (0.1 M Tris, pII 7.2) Triton X-100 Tergitol NPX Tergitol 15-S-9 Brij 58 Brij 35 Nomdet Pdg Lubrol WX Lubrol MOA Antarox BL-225 Antarox BL-240 Tween 20 Tween 40 Tween 60 Tween 80 Deoxycholate Phospholipase A Cetylpyridinium Bromide
2.94 12.8 12.5 5.1 10.9 -6 8.9 9.3 2.1 5.8 6.0 2.3 3.4 0.0 6.7 0.3
Ratiob
Transferases in submaxillary gland Sd membrane
Glycoprotein:gal transferase CPw$
451
BIOSYNTHESIS
X
Ratioh
Polypeptidyl:gaWAc transfera%? CPm$J
X
Glycoprotekgal transferase
Ratiob
“P;$F/
X
R&oh
1.0
3.9
1.0
3.7
1.0
5.8
1.0
4.4 4.3 1.7 3.7
45.4 48.2 44.8 56.5
11.6 12.3 11.5 14.4 -
3.1 3.2 0.7 -
73.3 56.5 14.9 -
18.7 14.4 3.8 -
2.0 2.1 0.8 1.2 0.0 2.3 0.1
32.5 20.7 14.7 19.2 1.0
34.3 36.9 27.8 29.1 20.0 33.0 29.6 4.9 26.1 15.6 12.0 13.4 10.6 14.3 9.8 8.3 1.1
9.3 10.0 7.5 7.9 5.4 8.9 9.0 1.3 7.0 4.2 3.3 3.6 2.9 3.9 2.6 2.2 0.3
42.4 45.6 49.5 38.3 27.7 46.5 49.6 5.7 41.1 41.6 25.0 12.6 9.8 14.0 2.2 11.6 2.9
7.3 7.9 8.5 6.6 4.8 8.0 8.5 1.0 7.1 7.2 4.3 2.2 1.7 2.4 0.4 2.0 0.5
8.3 5.6 3.8 5.0 0.25
0 Detergents Tritons W30 and 770 and sodium dodecyl sulfate tivities are close to 0. b Ratio refers to the activity (+ detergent/ - detergent). c These marks refer to activities not measured.
are not listed
becanse the resulting
ac-
452
HAGOPIAN
membrane preparation of HeLa cells, the maximum increase in specific activity was about four times greater for the glycoprotein: galactosyl than the polypeptidyl: Nacetylgalactosaminyl transferase. Bovine submaxillary membrane preparations (4S), when extracted with detergents, released both glycosyl transferases; the maximum increase in specific activity in this case was approximately S-lo-fold and compares with the HeLa cell preparations where a 4-l&fold increase was observed. As in HeLa cells, the detergents most effective for the release of galNAc transferase were Tergitol NPX and Triton X-100. The detergents Nonidet P40 , Lubrol WX, Brij 58, Tergitol 15-S-9, and Antarox BT,225 were 70-90 % as effective as the Tergitol NPX. The Tween series, Antarox BL-240, and Brij 35 solubilized approximat’ely 3054% as much enzyme activity as the best, detergent. Lubrol MOA and Triton 770 produced no effect over the Tris buffer while Triton W30 and sodium dodecyl sulfate totally inactivated the transferase. Phospholipase A and the ionic detergent deoxycholate had about 25 % the effect of Tergitol NPX. The best detergents for the release of the galactosyl transferase from St submaxillary membranes were Tergitol 15-S-9 and Lubrol WX. Also very effective were Nonidet P4,, (94% of Tergitol 15-S-9), Tergitol NPX (93 %), Triton X-100 (86 %), Ant,arox BL-240 (85 %), and BL-225 (84 %). Tritons W30 and 770 and SDS inactivated the galactose transferase while all other detergents used either had no effect or were less than 50% as effective as the best detergent.
The e$ect of the detergents on the solubilixed and puri$ed N-acetylgalactosaminyl transferase. In a preliminary step in the purification process
of the galNAc
transferase,
the
bovine submaxillary glands are extracted with Triton X-100 (14). This process releases the enzyme in soluble form and further purification is achieved by differential centrifugation and gel filtration (80-fold). To test the effect of the various detergents on the purified enzyme, the Triton X-100 already present was removed by gel filtration by Sephadex G-100 column chromatog-
AND EYLAR
III 0.8 2 0.6 ?i 6
0.4 l$
5
10
20
15 Tubs
25
30
I
0.2
Number
FIG. 2. Separation of purified polypeptidyl: galNAc from Triton X-100 by Sephadex G-100 column chromatography. The fractions were eluted with 0.05 M Tris buffer, pH 7.2. Each fraction was assayed for polypeptidyl:galNAc transferase activity (0-O). The optical density at 280 rnp of each fraction was also measured ( l --0). TABLE
III
THE EFFECT OF THE DETERGENTS ON THE TOTAL ACTIVITY OF THE PURIIVED POLYPEPTIDYL: galNAc TRANSFERASE ISOLATED FROM BOVINE SUBMAXILLARY GLANDS Detergent
Cpm/ml x 10-a
None (0.1 M Tris, pH 7.2) Triton X-100 Tween 20 Tween 40 Tween 60 Tween 80 Nonidet PRO Brij 58 Tergitol 15-S-9 Tergitol NPX Lubrol MOA Lubrol WX Triton 770 Triton W30 Sodium dodecyl sulfate Deoxycholate
14.7 20.4 14.0 15.7 11.2 12.3 10.1 14.8 10.6 12.2 14.6 13.0 3.0 0 0 0
raphy. Figure 2 shows the separation of the enzyme from the Triton X-100 detergent. The tubes containing the transferase were combined, lyophilized, and dialyzed against .005 M Tris buffer, pH 7.2. Table III shows the effect, of the various detergents on the purified polypeptidyl: galNAc transferase.
GLYCOPIIOTEIN
All the nonionic detergents had no deletcrious or advantageous effect on the enzyme activity when compared to the control, in which case no detergent, but Tris buffer, was added. On the other hand, the ionic detergent,s sodium dodecgl sulfate, sodium deoxpcholate, Trit,on W30, and Triton $70 tot,ally inactivated t’he galP\‘Ac transferase.
HIOSYNTHEYIS
453
bmnes of the HeLa cells (&) and the membrane fraction (S,) of the submaxillary tissue is demonstrated by both the yield and degree of purificat,ion (Table I). The main finding of t,his study is that regardless of the type of membrane in which t,he glgcosyl transferases are found, they appear to be intimately bound within the membranous framework; disruption of the DISCUSSION membranous structure is necessary for full expression of activity. From SO-95% of The object of this study was to investigat,e the total enzyme activities are masked in the dissociation of glycosyl transferases from their membrane environment where they are the isolat,ed membranes compared to the opintimat,ely bound. E’or this purpose, two timal conditions produced by the nonionic glycoprotein: glycosyl t)ransferases appeared detergents. This finding suggests that the in situ localization of the transferases as appropriate: the polypeptidyl:galNAc transferase, which is responsible for synthesis of part of the membrane structure represents an important aspect of their in vivo functhe protein-carbohydrate linkage involving tion. The data also suggest that nonpolar hydroxyamino acids linked glycosidically binding plays an important role in the to N-acetylgalactosamine (l-2), and the gslactosyl transferase which is specific for interaction of the transferases with their membranous environment. It is of interest synthesis of the /3-n-galactosyl-(M-Nin this regard that in both types of memucetylglucosamine linkage in glycoproteins branes (fraction S1 from HeLa or fraction (I&19). Although these two enzymes show a Sb from submaxillary glands) the transhigh specificity for their receptors, the resultant’ glgcoprotein product may vary de- ferases appear to respond similarly to the pending on the choice of tissue. For example, various detergents. The nonionic detergents Triton X-100, Tergitol NPX and 15-S-9, in the HeLa cells these enzymes are involved Nomdet Pqg , and Lubrol WX are most efin the assembly of membrane glycoproteins, fect,ive in general and lead to maximal acwhereas in t,he submaxillary gland they part,icipate in the synt’hesis of secreted tivity. In general, the detergents show the same relative effect on each enzyme from submaxillary glycoprot’eins. For comparathe two types of membranes; the Tween tive purposes, therefore, HeLa cell transferases were chosen for comparison with the series lead to intermediate activities and results with sodium analogous submaxillary transferases. As seen complete inhibition dodecyl sulfate and Triton W30, which are from Table I, the cellular localization of negatively charged molecules. t.hese enzymes differ depending on the With respect t,o the action of the various choice of tissue; in HeLa cells, they are found in the smooth internal membranes as detergents, it is of interest to compare detergent structure with the activities found part of a multienzyme group of transferases for the transferases. The nonionic deter(9), whereas in the submaxillary tissue they gents are of t’he polyoxyethylene type. The are found near the 40-50% sucrose intermost effective det,ergents, such as Triton face. It is possible in the latter case that X-100, Tergitol NPX, and Nonidet PbOare the glycosyl t’ransferases are located in the of the polyoxyethylene octylphenol type; plasma membranes which migrate to t.his slightly less effective are the linear primary position during the isopyenie centrifugaalcohol polyoxyethylene types such as tion. Such is the case for the collagen: Antarox BL-225 and Brij 35. The Tween glucosyl transferase found in the HeLa cells (9), which is involved in synthesis of detergents, which are generally less than collagen, a secreted glycoprotein. It is 50% as effective as the octylphenol series, noteworthy that the localization of the are polyoxyethylene sorbitan monofatty glycosyl t,ransferases in the smooth mem- acids. Thus, the grouping to which the
polyoxyethylene chain is attached has considerable influence on the ability of the detergent to disrupt nonpolar membrane structures and solubilize the glycosyl transferases. Those detergents containing the alkylphenol grouping appear to be most effective. The influence of charged groups on the basic nonpolar detergent molecule are clearly illustrated by Triton W30 and Triton 770 which are sulfonated or sulfated polyoxyethylene alkylphenols, respectively. Both result in complete inhibition in nearly every case. The other negatively charged molecules, such as sodium dodecyl sulfate and deoxycholate, also give a high degree of inhibition. The inhibitory action of charged detergents is not restricted to the negatively charged molecules, but also is found with cetylpyridinium bromide, a detergent with a positive charge. In addition to detergents, membrane dissociation with phospholipase A also resulted in partial release of the transferases. With the N-acetylgalactosaminyl transferase from HeLa cells, the phospholipase A treatment increased the activity nearly 5-fold. In view of the action of the phospholipase A, which leads to release of fatty acid from the phospholipids of the membrane, it appears that nonpolar forces play a significant role in membrane substructure. This result is comparable with the recent subunit concept of membrane structure in contrast to the Davson-Danielli unit membrane hypothesis (16) ; a similar conclusion was derived by Lenard and Singer (17) using erythrocyte membranes and phospholipase C. In evaluation of the initial influence of detergents on the transferases, the resultant activity appears to depend on two factors, solubilization and inhibition. In order to answer questions concerning the activation or inhibition of detergents on the glycosyl transferases as well as the possible nonpolar stabilization of these enzymes once separated from their membranous environment, polypeptidyl: N-acetylgalactosaminyl the transferase was subjected to gel filtration in order to remove the detergent. Using the polypeptidyl: N-acetylgalactosaminyl transferase purified from submaxillary gland membranes it is evident that the enzyme
does not require the presence of detergent for full activity. Upon removal of the Triton X-100, the enzyme activities were approximately the same with or without detergent. Although the adsorption of a low level of Triton X-100 cannot be discounted, it appears that most of the detergents neither inhibit nor activate this enzyme. It can be concluded, for example, that the failure of Tween 80 to give full activity is due to inefficient solubilization. It is apparent, however, that the complete inactivity in the presence of negatively charged detergents, such as Triton W30, sodium dodecyl sulfate, and sodium deoxycholate, is due to inactivation of the enzyme rather than a lack of solubilization. It is of interest that these polar detergents, which are commonly used in biochemical studies, lead to inactivation, whereas the nonpolar detergents are without influence generally. Furthermore, the enzyme prepared in this manner by gel filtration is stable to lyophilization and freezing even over a period of several months and, thus, does not require detergent stabilization. It appears, therefore, that the main effect of the nonpolar detergents on the membranes with respect to the glycosyl transferases is release and solubilization. The detergents are not required for activation or stabilization. It cannot be decided from this study if the different effects produced by the ionic and nonionic detergents is due to a difference in micellar structure, direct inactivation of the enzymes, or to some other factor. The transferases of this report contrast with those found by Roseman and co-workers which are not membrane bound, but exist normally in a soluble form in goat colostrum (E-20). In a preliminary study, Schachter and McGuire (21) reported on a glycosyl transferase from porcine submaxillary gland that is particulate bound. ACKNOWLEDGMENTS We are indebted to Mr. Jesse Jackson and Mr. Roger Birosel for valuable technical assistance and to Mr. Donald Wegemer and Mrs. Chris Law for growing the HeLa cells used in this work. We also thank Dr. Jonas Salk for helpful advice and encouragement.
GLYCOPROTEIN
BIOSYNTHESIS
REFERENCES 1. SARCIONE, E. (1964).
J., J.
Biol.
Chem. 239, 1686
2. MOLNAR, J., ROBINSON, G. B., AND WINZLER, R. J., J. Biol. Chem. 240,1882 (1962).
3. O’BRIEN, P. J., CANADY, M. R., HALL, C. W., AND NEUFELD, E. F., Bioehem. Biophys. Acta 117, 331 (1966). 4. SIMBIN, J. L., AND JAMIESON, J. C., Biochem. J. 103, 38P (1967). 5. LA~FORD, G. R., AND SCHACHTER, H., J. Biol. Chem. 241, 5408 (1966). 6. LI, Y., LI, S., AND SHELTER, M. R., J. Biol. Chem. 243, 656 (1968). 7. COOK, G. M. W., LAICO, M. T., AND EYLAR, E. H., Proc. Natl. Acad. Sci. U. S. 64, 247 (1965). 8. BOUCHILLOUX, S., AND CHEFTEL, C., Biochem. Biophys. Res. Commun. 23,307 (1966). 9. HAGOPIAN, A., BOSMANN, H. B., AND EYLAR, E. H., Arch. Biochem. Biophys. 128,387 (1968). 10. FISZER, S., AND DEROBERTIS, E., Brain Res. 6, 31 (1967). 11. EYLAR, E. H., CACUM, J. F., HAGOPIAN, A.,
12. 13. 14. 15.
16. 17. 18.
19.
20.
21.
455
AND LAICO, M. T., Abstr. IV Intern. Congr. Biochem. (1967). CACCAM, J. F., AND EYLAR, E. H., in preparation. LAICO, M. T., AND EYLAR, E. H., Federation Proc. 26, 2252 (1966). HAGOPIAN, A. AND EYLAR, E. H., Arch. Biothem. Biophys. 129,515 (1969). BOSMANN, H. B., HAGOPIAN, A., AND EYLAR, E. H., Arch. Biochem. Biophys. (1968). In press. KORN, E. D., Science 163,149l (1966). LENARD, J., AND SINGER, S. J., Science 169, 738 (1968). BARTHOLOMEW, B., JOURDIAN, G. W., AND ROSEMAN, S., Abstr. 6th Intern. Congr. Biothem. IUB 32,503, vi-i (1964). MCGUIRE, E. J., JOURDIAN, G. W., CARLSON, D. M., AND ROSEMAN, S., 1. Biol. Chem. 240, PC 4112 (1965). JOHNSTON, I. R., MCGUIRE, E. J., JOURDIAN, G. W., AND ROSEMAN, S., J. Biol. Chem. 241, 5735 (1966). SCHACHTER, H., AND MCGUIRE, E. J., Pederation Proc. 27, 345 (1968).