Eur. J. Biochem. 124, 383-388 (1982) 0 FEBS 1982

Removal of Wheat-Germ Agglutinin Increases Protein Synthesis in Wheat-Germ Extracts

Abraham K . ABRAHAM, Svein KOLSETH, and Alexander PIHL

Norsk Hydro's Institute for Cancer Research, Oslo; and Department of Biochemistry, University of Bergen

(Received August 26, 1981 /January 28, 1982)

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin- treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied. indicated that addi- tion of wheat germ agglutinin reduced the amount of available ribosomes.

Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglu- tinin was not released by acid treatment.

The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis. at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.

Extracts from raw wheat germ have been widely used for the characterization of the translation products of eukaryotic messengers. The wheat germ system is inexpensive and has the advantage that the endogenous protein synthesis is very low. Recently it has been shown that in the presence of poly- amines it is capable of also translating large messengers [l - 31. However, in our experience the efficiency of the system varies considerably from batch to batch. The possibility was con- sidered that this might be due to the presence of varying con- centrations of some inhibitor. We therefore subjected the extract to chromatography on a column of chitin which has a strong affinity for wheat germ agglutinin [4]. Here we report that chitin affinity chromatography increases the pro- tein-synthesizing ability of wheat germ extracts and we present experiments designed to elucidate the mechanism underlying the observed effect.

MATERIALS AND METHODS

Chem iculs

Chitin (from crab shells, practical grade), spermidine, wheat germ agglutinin, GTP and ATP were obtained from Sigma Chemicals (St Louis, MO, USA). Creatine phosphate and creatine phosphokinase were purchased from Calbio- chem AG (Switzerland). High-specific-activity I4C-labelled amino acid mixture (CFB 104) and [I4C]formaldehyde were obtained from The Radiochemical Centre (Amersham, Eng- land).

A6hrrviuliori. Hepes, 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid.

Prepmation of Chitin Column

The chitin column (1.5 x 0.4 cm) was prepared and washed as described earlier [ 5 ] and finally with the elution buffer (20 mM Hepes, pH 7.6, 120 mM KCI, 5 mM MgC12, 1 mM dithiothreitol) used for the preparation of wheat germ extract.

Wlieal Germ Extracts

Extracts of raw wheat germ (Niblack, Rochester, USA) were prepared, essentially as described by Roberts and Pater- son [6], with the following modifications: no thiol was added to the extraction buffer and the elution buffer contained 1 m M dithiothreitol. In a typical experiment 5 p of raw wheat germ was ground with 5 g of glass powder and 15 rnl of extraction buffer at 2°C. After the centrifugation step, the extract was passed three times through a chitin column (0.5 x 1 cm) at a flow rate of 1 ml/min, then through a molecular sieve column as described [6] and centrifuged in a Sorvall SS 34 rotor for 10 min at 15000 rev./min. The supernatant was diluted with elution buffer to a concentration of 100 AZbo unitsiml. Small aliquots were frozen and kept stored in liquid nitrogen. Con- trol extracts were prepared in excatly the same way, except that the affinity chromatography was omitted.

Purification of' Rabbit Glohin m R N A

Rabbit globin mRNA was isolated by oligo(dT)-cellulose chromatography [7] and purified further by sucrose gradient centrifugation on a 5-20% linear gradient in 20 mM Tris- HCI (pH 7.2) at 40000 rev./min in a SW-40 rotor for 16 h.

Pulywrylunii& Gei Elecfrophowsis

Polyacrylamide gel electrophoresis in dodecyl sulfatc was carried out in 0.7-mm thick, 14 x 28 cm slab gels, consisting

3x4

of 19.5 ?" acrylamide and 0.5 "1" bisacrylamide, using Laemm- li's buffer [8], as described earlier [2].

Pro t oin Sj-n t liesis in v it r o

Polypeptide synthesis directed by rabbit globin mRNA was measured as described earlier [9] with slight modifications. The reaction mixture (100 pl) contained 40 mM Hepes, pH 7.7, 1.5 mM magnesium acetate, 0.66 mM spermidine, 50 pM CaCI2. 66 mM KCI, 1 mM ATP, 0.2 mM GTP, 1.0 mM dithiothreitol, 8 mM creatine phosphate, 2 units of creatine phosphokinase, 1 .O pCi of ''C-labelled amino acid mixture containing 14 amino acids (CFB 104), six unlabelled amino acids (10 pM each) and the indicated amounts of mRNA and wheat germ extract.

Separation of' Ribosomal Suhunits

Wheat germ extract was incubated with 1 mM puromycin and 1 mM GTP at 30-C for 5 min. After incubation the KCI concentration was increased to 0.5 M and the mixture was layered onto a 10 - 30 sucrose gradient in the buffer con- taining 40 mM Hepes, pH 7.7, 1.5 mM magnesium acetate, 1 mM dithiothreitol, 0.66 mM spermidine and 0.5 M KCI, and centrifuged at 40000 rev./min in an SW-40 rotor for 4.5 h .

Prcyuratiotz of Ribosomes and Postribosoniul Fraciiotzs

The KCI concentration of the wheat germ extract, prepared as described above, was adjusted to 0.5 M with 2 M stock solution. The extract was layered on top of 1 nil of elution buffer [6] containing lo;< sucrose and centrifuged at 50000 rev./min in an SW-50.1 rotor for 2 h. The pelleted ribosome fraction was rinsed with elution buffer and suspended in the same buffer at a concentration of 9 Azho unitsjml. The super- natant fraction was passed through a Sephadex G-50 column, equilibrated with elution buffer to remove excess KCI and sucrose. Ribosomes and supernatant fractions were used immediately after separation.

Reduction and Carhoxymethylation of Wheat Grrm Agglutinin

Wheat germ agglutinin (2 mg) was reduced with a 500-fold molar excess of dithiothreitol in 50 mM Tris/HCI buffer (pH 7.6) and carboxymethylated with a 2000-fold excess of sodium iodoacetate according to Erni et al. [lo]. Excess of iodoacetate and low-molecular-weight compounds were re- moved by two successive gel filtration steps, using a Sephadex (3-50 column (0.9 x 40 cm) equilibrated with 50 mM Tris/HCl buffer.

Prcpirution of ''C-Lahelled Wheat Germ Agglutinin

Native agglutinin and carboxymethylated agglutinin were labelled with ['4C]formaldehyde according to Rice and Means [I I ] in 0.2 M borate buffer. Labelled proteins were thoroughly dialyzed before use.

Hemagglutinating Activity of Wlieat Germ Extract

The hemagglutinating activity of the wheat germ extract was determined by a serial dilution assay in microtiter plates [lo], using a 2% suspension of washed human AB Rh' red

blood cells. The concentration of agglutinin in the extract, using Sigma wheat germ agglutinin as standard, was found to be in the range 40-85 pg inl.

RESULTS

ELfi2c.t of Chitin Coliinitz Clzromutogrupplij~ on Protein - Sjw tlwsiz in;: A h ility ?f Wli ea t Germ E.Y t rac ts

Protein synthesis in untreated and chitin-treated wheat germ extracts is demonstrated in Fig. 1 and 2. In the untreated extract the rate of protein synthesis declined strongly after about 30 inin and almost levelled off after 60 min. In contrast. in the chitin-treated extract, the rate of protein synthesis was higher and the synthesis proceeded almost linearly for 60 min. Since the N-acetyl-D-glucosamine polymer, chitin, has a strong affinity for wheat germ agglutinin [12], purified agglu- tinin was added back to the chitin-treated extract. I t is seen (Fig. 1) that 100 pglml of wheat germ agglutinin reduced the rate and extent of protein synthesis to about the levels seen in the untreated extract. This amount is considerably larger than that removed from the extract by the chitin treatment. From the hemagglutination assay it can be calculated that the amount removed (40 -- 85 pglml extract) corresponds to about 12-26 pg of wheat germ agglutinin/ml of protein syn- thesis mixture. The significance of this apparent discrepancy will be discussed below.

Addition of wheat germ agglutinin to untreated extracts gave varying results depending on the preparation. Thus, in untreated extracts which initially had a low protein-synthesiz- ing ability, addition of wheat germ agglutinin had little or no inhibiting effect, whereas in preparations with higher activity, addition of agglutinin inhibited the protein synthesis appreci- ably (data not shown). Altogether, these results indicate that wheat germ agglutinin inhibits protein synthesis and that the variable ability of wheat germ extracts to support protein synthesis may be related to different contents of wheat germ agglutinin.

Mechanism of the Inhibiting effect of Wheat Gcrm Agglutinin on Protein Synthesis

To elucidate the mechanism of the inhibition by wheat germ agglutinin, the protein synthesis was studied under con- ditions when either the amount of wheat germ extract or the concentration of added messenger was varied. It is seen (Fig. 3 A) that when the amount of messenger was kept con- stant, addition of increasing amounts of extract reduced strongly the inhibition of protein synthesis by wheat germ agglutinin. This indicates that the agglutinin interacts with a component(s) in the extract, rendering this limiting. Conver- sely, when the extract concentration was kept constant (40 A260 unitsiml), the inhibition of protein synthesis by wheat germ agglutinin increased with increasing concentration of added messenger (Fig. 3 €3). The results show that the inhibi- tion of protein synthesis by wheat germ agglutinin depends strongly on the ratio of extract to messenger and they indicate that in the presence of wheat germ agglutinin some compo- nent(s) in the extract becomes limiting for the protein syn- thesis.

To study which component(s) in the extract is affected by wheat germ agglutinin, we measured the inhibition of protein synthesis at a fixed messenger concentration and increasing concentrations of either supernatant factors or isolated ribo- somes (Table 1). Under these conditions addition of ribo-

3x5

A

:J

I I

60

50

4 0 -

- - g

8

0

- 3 0 -

$ 20- - Q

-

I 10 20 30 40 50 80

Time (rnln)

-

-

Fig. 1. Protein synthe.~is in chitin-treated and unireuted whear germ extracts. Protein synthesis was measured as described under Materials and Methods, using 2 Az60 units of extract and 0.02 unit of rabbit globin mRNA. Trichloroacetic-acid-insoluble radioactivity was deter- mined in 2 0 4 samples at different time intervals. (.) Untreated extract; (0) chitin-purified extracts; (A) chitin-purified extract incubated in the presence of 100 pg/inl of wheat germ agglutinin

Fig. 3

/// 0

l o t n l I I I

" 0 0.2 0.4 0.6 0.0 mRNA (A,,, unitsiml)

60

50

- - g 40 0

s - 30 P e - .: 20 -

10

0 I 40 80 120 160 200

Agglutinin (pglml)

Fig.2. Efyect of wheat germ agglutinin on protein synthesis by (,hitin- puri jkd wheat germ exlracts. Protein synthesis was measured as described under Materials and Methods, using chitin-purified wheat germ extract, and increasing concentrations of agglutinin. The reaction mixture con- tained 2 ,4260 units of wheat germ agglutinin and 0.02 AZhO unit of mRNA. After preincubation for 5 min in the absence o f amino acids, protein syn- thesis was started by adding the amino acids. The incubation was stopped after 60 min. The data are expressed as the inhibition as a percentage of the control, incubated in the absence of agglulinin

hibition ofprotein synthesis by wheat germ ugghtinin at d$]krent ratios ofextruct to mRNA. Protein synthesis in a chitin-treated er act was measured as in Fig. 1. The incubation lasted for 1 h, and the concentration of agglutinin was 100 pg/ml. The inhibition is expressed as a percentage of the control samples, incubated in the absence of agglutinin. (A) The concentration of extract was varied, as indicated, using a fixed mRNA con- centration of 0.2 Azoo unit/ml. When the extract concentration was increased from 5 A m units/ml to 60 AZh0 units/rnl, the absolute values of amino acid incorporation in the control samples increased from 1.4 x 104 counts/min to 3.9 x lo4 counts/min. (B) The mRNA concentration was varied, as indicated, using 40 A z ~ ~ units/ml of extract. When the mRNA concentration was increased from 0.2 to 0.8 A260 unit,'ml, the absolute values in the control samples increased from 3.2 x lo4 counts/min to 9.8 x lo4 counts/min

somes decreased markedly the inhibition, whereas increasing concentrations of supernatant had no such effect. The results suggest that wheat germ agglutinin inhibits protein synthesis by interacting with the ribosomes.

To study the nature of the interaction of the agglutinin with ribosomes, chitin-treated extracts were incubated with 14C-labelled wheat germ agglutinin and the mixtures were subjected to sucrose gradient density centrifugation. It is seen that when intact wheat germ agglutinin was used (Fig.4A), a considerable fraction of radioactivity was associated with the monosome and polysome region, indicating that the agglutinin is bound to the ribosome fraction. In contrast, when reduced and carboxymethylated agglutinin was added, essentially no radioactivity was present in the polysome and monosome region (Fig. 4B).

In separate experiments not illustrated, it was found that the reduced and carboxymethylated agglutinin which did not bind to the polysomes, also failed completely to inhibit protein synthesis. This provides evidence that the binding of wheat germ agglutinin to the ribosome fraction may in fact account for its inhibiting effect on protein synthesis.

To investigate the possibility that the agglutinin might be bound to ribosome-associated factors rather than to ribo- somes as such, chitin-treated wheat germ extract was incu- bated with labelled wheat germ agglutinin in the presence of puromycin and GTP and high concentrations of salt. Under these conditions, ribosome-associated factors are released and the ribosomes dissociate into their subunits. Sucrose gradient density centrifugation of the reaction mixturc revealed (Fig. 5) that labelled wheat germ agglutinin sedi-

Table 1. Pvofeiii syn/llt~.si,s inhihition /J,I iihrat gcwn agglu/inin in th t . prr.c- c w c ~ ' of increcrsing arn0unt.s of ,supcmuraizt ,fuctor.s and ri11osonw.s Protein synthesis was measured as desci-ibed under Materials and Methods. using 2 AZhll units of chitintreated wheat germ exti-iict and 0.03 AZho unit of rabbit globin mRNA. The concentration of agglutinin. when present, was 80 pgiml. After a 60-min incubation at 30'C. 20-pI samples were taken in duplicatc for radioactivity meaSurenlents

Additions 10- x Radioactivity Inhibition

agglutinin chitin- present treated

extract

0 I AZbo unit counts,min , <,

None 16.9 28.5 41

Ribosomes 0.3 19.9 30.1 34 0.6 24.8 31 .0 1-0 0.9 26.8 31.1 14

Supcrnatant 0.4 16.7 28.0 40 0.8 16.8 28.2 40 1.2 17.2 28.6 40 2.0 17.8 29.2 30

0 ;!I I , I I

I I

128.0

t 32 .0 5

c c 3 0 0 - E

8.0 5 3 0 - m m U - -

2.0 ; - 0

0.5

- 0

0 8 16 24 0 8 16 24 32 Fraction number

Fig. 4. Su iwse ilmsity gradimt of iv/7cul gtt'n? c'xfrrrctr i t l ~ U h U t d it/? luhrllcd intac~ whuat germ cigglutinin or with rrctucd and c ,~iho.~j , - mrthj~loted ii.hea/ g:crr?z agglutinm. The reaction mixture (100 pi) con- taining 2 AZhO units of extract and 10 pg (15000 countsimin) of I4C- labclled intact or reduced and carboxymethylated wheat germ agglutinin was incubated for 10 min at 28 'C under conditions used for protein syn- thesis. The samples wcre layered on top of 15-35yc1 linear sucrose gradients in 40 mM Nepes buffer (pH 7.7) , containing 70 inM KCI. 1.5 niM MgC12 and 0.66 mM spermidine, and centrii'uged at 50000 rev.; min for 45 inin in a Beckman SW 50.1 rotor. (A) Intact agglutinin; (B) reduced and carboxymethylated agglutinin

mented together with the 60-S subunits and to a small extent also with the 40-S subunits. The results demonstrate that wheat germ agglutinin in fact binds to ribosomes free of associated factors. This binding was not reduced in the pres- ence of 100 mM N-acetyl-o-glucosaniine in the gradient, and the bound radioactivity was stable to treatment with trichloro- acetic acid precipitation (data not shown).

5 10 15 2 0 Fraction number

Fig. 5. Si4cro.s~ c lmsi t j~ gt'~idiei1t.c. .seprrr~tion of rihosornal .suhunils iiicuhtrrrd iritii imhcdicd i i ~ h c u r gcwi agglufinin. The reaction mixture (200 pi) con- taining 2 AZbo units of extract, 1 mM puromycin and 1 m M GTP in elution buffer [4] was incubated for 5 min at 30"C. The KCI concen- tration was increased to 0.5 M and the sample was incubated for 10 niin at 30°C with 20 pg (30000 countsiniin) of 'SC-labelled wheat germ agglutinin, and the subunits were separated as described under Materials and Methods

Merhatiisn? of Bindiiig of Wlicur Germ Agghtiniri to Ribosomes

Wheat germ agglutinin binds to N-acetyl-D-glucosamine and to sialic acid, and it has been found that a large excess of these sugars can counteract the binding of wheat germ agglu- tinin to cell surface glycoconjugates [14]. Addition of 1 mM sialic acid and as much as 100 mM N-acetyl-D-glucosamine did not counteract the ability of the agglutinin to inhibit protein synthesis (Table 2), indicating that the binding of agglutinin to ribosomes does not involve its sugar binding sites.

Since ribosomes possess exposed disulfide and sulfhydryl groups on their surface [13], the possibility was considered that wheat germ agglutinin may be bound to ribosomes through mixed disulfide formation. The effect of thiols on protein synthesis inhibition by agglutinin was therefore studied. I t was found (Table 2) that increasing concentrations of thiols diminished the inhibitory effect of the agglutinin. At the highest concentration of thiols used the inhibition of pro- tein synthesis was diminished by about 50 "&

DISCUSSION

The present finding that chromatography of wheat germ extracts on a chitin column increases both the rate and extent of the protein-synthesizing activity of the extract is of interest from several points of view. In the first place, the results inay increase the usefulness of this system. Here the effect of chitin chromatography was demonstrated with the relatively short globin messenger. However, wheat germ extracts supple- mented with polyamiiies also translate long messengers. such as tobacco mosaic virus RNA [2], and we have found (data not shown) that chitin treatment of'the extract enhances the trans- lation of this messenger a s well.

Secondly, the mechanism of the enhancing effect of chitin treatment is of some interest. Evidence is presented here that

187

Table 2. /<f/iw of' . S U ~ U ~ . S n d tliiols on protcin .syr21liesis inhihition hJ' wheat grnn agglutinin Assay conditions were as described in Table 1 . The concentration of agglutinin. when present, was 100 pg,'ml. All samples contained 1 mM dithiothreitol in addition to the indicated concentrations of thiols

Additions Concn x Radioactivily Inhibition

turing agents. Since thiols are always present under the condi- tions used for protein synthesis, at least some of the disulfide groups of the wheat germ agglutillin itre probably reduced, The disulfide and thiol groups ofthe agglutinin may therefore interact with thiol and disulfide groups, respectively, on the surface of the ribosomes to form mixed disulfdes by the fol-

~~~~ ~ lowing reversible reactions: agglutinin chitin- absent treated

extrac1

0 I mM c o u n t s h i n I 0

None -

N-Acetylglucos- aminedimer 5 (chitobiose) 100

Sialic acid 5

Mercaptoet hanol - 2, 4 6 X

Dithiothreitol 1 2 3 5

27.7

28.1 24.8

26.9

30.8 32.3 31.6 24.2

30.2 33.9 34.6 24.S

13.2 52

13.7 50 12.9 4s

12.8 52

20.6 33 23.4 28 24.2 23 18.5 24

19.5 35 22.8 32 23.2 33 16.0 26

it is due, at least in part, to the removal from the extracts of wheat germ agglutinin which is able to inhibit protein syn- thesis by interacting with the ribosomes. Thus, addition of purified agglutinin to chitin-treated extracts inhibited protein synthesis and the agglutinin was shown to bind to the ribo- somes. I t is significant that reduced and carboxymethylated wheat germ agglutinin which failed to bind to the ribosomes, was unable to inhibit protein synthesis.

The apparent discrepancy between the concentration of wheat germ agglutinin in untreated extracts and the concen- tration of commercial wheat germ agglutinin required to inhibit protein synthesis to the level observed in untreated extracts, may have several explanations. In the first place, since the ability ofthe wheat germ agglutinin to inhibit protein synthesis does not involvc its sugar binding sites, it may be unrelated to its agglutinating ability. Secondly, it is possible that the chitin chromatography also removes other inhibitors of protein synthesis from the extract. In fact, we have recently obtained preliminary evidence that added messenger is more stable in chitin-treated than in untreated extracts. Thus, a greater fraction of added mRNA could be recovered from chitin-treated than from untreated extracts, as determined by hybridization to complementary DNA, and a larger percent- age of the radioactivity incorporated into polypeptides could be recovered in completed globin molecules (data not shown).

The mechanism of the binding of wheat germ agglutinin to ribosomes apparently does not involve its sugar binding sites, since addition of excess N-acetyl-D-glucosamine was unable to counteract the protein synthesis inhibition. How- ever, increasing concentrations of thiols did counteract the inhibitory effect on protein synthesis. This is consistent with the possibility that wheat germ agglutinin is bound to the ribosomes through mixed disulfide formation, as discussed below.

It has recently been shown [lo] that wheat germ agglutinin can be completely reduced by thiols in the absence of dena-

/4 Wheat germ agglutinin + HS-ribosome ' S

+ Wheat germ agglutinin - S - S-ribosome. ( 1 ) \

SH

s\ S /

Wheat germ agglutinin-SH + 1 ribosome

Wheat germ agglutinin- S- S-ribosome. (2) I SH

This mechanism is supported by the finding that the ribo- some-bound wheat germ agglutinin was stable to acid treat- ment which dissolves wheat germ agglutinin into monomers and should dissociate any non-covalcnt interaction. Acid treatment will freeze the equilibria in ructions 1 and 2, as i t removes the nucleophilic R S groups [ 151. Binding of wheat germ agglutinin to ribosomes would bc expected to interfere with their interaction with the factor.; involved in protein synthesis.

As native wheat germ agglutinin possesses 16 disulfidc bridges and no free - SH groups [lo, 121. the binding of intact labelled wheat germ agglutinin to ribosomes observed herc probably involved primarily reaction 1. Obviously, in such exchange reactions several ribosomes may be bound to the same agglutinin molecule.

Thiols, such as dithiothreitol and niercaptoethanol, which are necessary componcnts of cell-free systems for protein syn- thesis, will interact with and split the mixed disulfides formed in reactions 1 and 2. This may explain thc fact that the inhibi- tion of protein synthesis by wheat germ agglutinin was incom- plete, even in the presence of high agglutinin concentrations and that increasing concentrations of dithiothreitol and mcr- captoethanol counteracted the inhibition by wheat germ agglutinin. The inhibition of protein synthesis by wheat germ agglutinin could not be completely abolishcd by thiols at concentrations which did not inhibit protein synthesis pc'r .YC.

It is therefore clear that a beneficial cffcct on protcin syn- thesis, comparable to that of chitin treatment, cannot be achieved merely by increasing the thiol concentration.

This work was supported by The Norwegian Cancer Society. Thc authors wish to thank Dr Sjur Olsnes for \aluable discussions and Dr H. B. Jensen for providing us with pretreated chitin. The skilful technical assistance of Mrs Tove Nesse and Mrs Mai-ita Johanscn is gralcfully acknowledged.

REFERENCES 1. Hunter, A. R., Farrell, P., Jackson, R. J . & Hunt, T. (1077) Eur. J .

2. Abraham. A. K . & Pihl, A . (1980) Eur. .I Uictciwnr. I06, 257-262. 3. Abraham, A. K . , Olsnes, S. & Pihl, A. (1979) FKBS Lct1. 101.

4. Bloch, R. & Burger. M . M . (1974) Bioc./rcni. Biophys. R ~ , Y . Conznzurr.

Biochanz. 75, 149- 157.

93-96.

58, 1 3 - 1 9.

388

5 . Jensen, H. B. & Kleppe, K. (1972) Eur. J . Biochem. 26, 305-312. 6. Roberts, B. E. & Paterson, B. M. (1973) Pro(. Natl Acad. Sci. USA,

7. Abraham, A. K. & Pihl, A. (1977) Eur. J . Biochem. 77, 589-593. 8. Laemmli, U. K. (1970) Nature (Loud.) 227, 680-685. 9. Abraham, A. K . & Pihl, A. (1978) FEBS Lett. 87, 121 -124.

11. Rice, R. H. & Means, G. E. (1971)J. Bid. Chem. 246,831-832. 12. Allen, A. K., Neuberger, A. & Sharon, N. (1973) Biochem. J . 131,

13. Nika, H. & Hultin, T. (1979) Biochim. Biophys. Actu, 579, 10- 19. 14. Monsigny, M., Roche, A. C.. Sene, C.. Maget-dana, R. & Delmotte.

70,2330-2334. 155-162.

10. Emi, B., De Boeck, H ., Loontiens, G. L. & Sharon, N . (1 980) FEBS F. (1980) Eur. J . Biochem. 104, 147-153. Lett. f20,149- 154. 15. Eldjarn, L. & Pihl, A. (1956) Acta Chem. Scand. 10, 1054-1055.

A. K. Abraham, Biokjemisk Institutt, Universitet i Bergen. Arstadveien 19, N-5000 Bergen, Norway

S. Kolseth and A. Pihl, Norsk Hydro's Institutt for Kreftforskning, Det Norske Radiumhospital, Montebello, Oslo 3, Norway