THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No . 22, Issue of ’ November 25, pp . 14217-14221.1984 Printed in U.S.A.

Site of Synthesis of the A+ and @ Subunits of the Na,K-ATPase in Brine Shrimp Nauplii*

(Received for publication, February 29, 1984)

James A. Fisher, Lee Ann Baxter-Lowe, and Lowell E. Hokin From the Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Developing nauplii (embryos) of the brine shrimp Artemia salina are an excellent model system for studying the biogenesis of the sodium- and potassium- activated adenosine triphosphatase (Na,K-ATPase). The nauplii exhibit a burst of Na,K-ATPase synthesis between 6 and 32 h of development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We have now determined the sites of synthesis of the a and /3 subunits of the Na,K-ATPase in developing A. salina nauplii. Mem- brane-bound and free polysomes were isolated from nauplii, and RNA was extracted from the polysomes. The polysomal RNA was translated in uitro in a rabbit reticulocyte lysate, and the translation products were immunoprecipitated by anti-subunit antisera. The im- munoprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Our data show that the a subunit precursor is synthesized on membrane-bound polysomes and the /3 subunit precursor is synthesized on free polysomes. In addition, the a subunit precursor appears as two separate peptides on sodium dodecyl sulfate-polyacrylamide gels, which suggests that the two a subunit forms seen in mature brine shrimp Na,K- ATPase are products of two distinct messenger RNAs. The /3 subunit precursor appears as a single discrete band, unlike the mature @ subunit, which appears as a diffuse band.

~~~ ~

The Na,K-ATPase’ of brine shrimp exhibits several inter- esting features that make it useful for studying developmental regulation as well as biosynthesis of the enzyme. (a) At early stages in development of brine shrimp nauplii (embryos), Na,K-ATPase activity is undetectable. At about 6 h of devel- opment, enzyme activity begins to rise, eventually peaking at 32 h. A peak of synthetic activity occurs between about 16 and 20 h (1,2). ( b ) The a subunit of the Na,K-ATPase appears as two molecular forms on SDS-PAGE. These have been

* This work was supported by Grant PCM-8120635 from the Na- tional Science Foundation, Grant HL16318 from the National Insti- tutes of Health, and Grant 140882 from the University of Wisconsin- Madison Graduate School Research Committee The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: Na,K-ATPase, sodium- and potas- sium-activated adenosine triphosphatase (EC 3.6.1.3); VRC, vanadyl ribonucleoside complex; NEM, N-ethylmaleimide; SDS, sodium do- decyl sulfate; PAGE, polyacrylamide gel electrophoresis; TMK, a buffer containing 50 mM Tris-HC1 (pH 8.5), 250 mM KCl, 10 mM MgCI,, and 1 Fg/ml cycloheximide; EGTA, ethylene glycol bis(0- aminoethyl ether)-N,N,N’,N’-tetraacetic acid; Hepes, 4-(2-hydrox- yethy1)-1-piperazineethanesulfonic acid.

designated al and ap for the slower and faster migrating forms, respectively. These two forms have similar primary- and possibly higher-order structures (1, 2) but differ in certain functional aspects, such as ouabain binding and phosphoryl- ation (4, 5). (c) The relative amounts of a1 and a2 change during development. At 14 h, the a l a 2 ratio is about 55:45. With increasing age, the amount of a2 gradually declines so that at 48 h the a1:a2 ratio is greater than 4 1 (1, 2).

As part of our ongoing studies of Na,K-ATPase structure and function, we have analyzed the mode of synthesis of the enzyme. In this paper, we have determined the site of synthe- sis of the a and fi subunits of the Na,K-ATPase of the developing brine shrimp Arterniu salina. The data presented here show that the a subunit is synthesized on membrane- bound polysomes but that the /3 subunit is synthesized on free polysomes. Transmembrane insertion of the two subunits would thus seem to be effected by different mechanisms. In addition, we show that the a subunit precursors are actually two distinct peptides; this indicates that a1 and a2 in the mature enzyme arise from distinct messengers rather than from differential modification of a single precursor.

EXPERIMENTAL PROCEDURES

Materials-Vanadyl ribonucleoside complex was either purchased from Bethesda Research Laboratories or prepared according to the method of Berger and Birkenmeier (6). Proteinase K was from Bethesda Research Laboratories. Micrococcal nuclease was purchased from Boehringer Mannheim. Methylmercuric hydroxide, supplied as a 1 M aqueous solution, was purchased from Alfa-Ventron. L - [ ~ ~ S ] Methionine was from Amersham Corp. Rabbit reticulocyte lysate was from Green Hectares, Oregon, WI. All other chemicals were pur- chased from Sigma.

Preparation of Polysomal RNA-Brine shrimp (A. salina) were reared as described by Peterson and Hokin (3). The artificial seawater was supplemented with 25 mg/liter streptomycin sulfate and 300 mg/ liter penicillin G (approximately 1600 units/mg). After 16 h of growth, the developed nauplii were collected on Miracloth TM (Calbiochem- Behring) and rinsed with distilled water.

Free and membrane-bound polysomes were isolated by a modifi- cation of the method of Ramsey and Steele (7). The collected nauplii were homogenized in TMK-B10 (TMK containing 0.44 M sucrose, 10 mM VRC, 1 mg/ml yeast tRNA, 1 mM EGTA, and 10 mM dithio- threitol) buffer with 10 strokes of a close-fitting Dounce homogenizer. Nauplii from 50 g of dry cysts were homogenized in 150 ml of buffer. The homogenate was filtered through two layers of sterile Miracloth and centrifuged for 5 min a t 1,700 X g. The post nuclear supernatant was centrifuged for 2.5 min at 1,600 X g and then for 5 min at 132,000 X g in a Beckman SW 27 rotor. The pellets were resuspended in TMK-B10, 1/4 volume of 100 mM VRC (relative to volume of TMK- BlO), and 1/9 volume of Triton X-100, homogenized in a Potter- Elvehjem homogenizer, and centrifuged for 10 min at 17,000 X g. To the supernatant was added 1/9 volume of 13% sodium deoxycholate (relative to the supernatant volume). The original supernatant (con- taining mostly free polysomes and some microsomes) and the deter- gent-treated supernatant (containing mostly released membrane- bound polysomes) were layered separately over 8 ml of TMK-C (TMK containing 2 M sucrose, 10 mM VRC, and 10 mM dithiothreitol) and

14217

14218 Site of Synthesis of Na.K-ATPase Subunits centrifuged for 22 h a t 132,000 X g.

The polysome pellets were resuspended in NET (50 mM Tris-HCI (pH 7.4), 100 mM NaCI, 0.5% SDS, and 20 mM EDTA) containing 0.5 mg/ml Proteinase K. After about 30 min of incubation a t room temperature (the time usually required to completely resuspend the polysome pellets), the polysomes were extracted four times with phenol/chloroform (1:l) and the extracted RNA was ethanol-precip- itated. The precipitated RNA was washed three times in 3 M sodium acetate and reprecipitated with 70% ethanol, 30% 0.2 M potassium acetate.

Preparation of Antibodies-@ subunit of brine shrimp Na,K- ATPase was purified as described by Peterson and Hokin (3). Briefly, fi subunit was purified by Bio-GelTM A-1.5m (Bio-Rad) chromatog- raphy and SDS-PAGE. f i subunit was eluted from the gel and emul- sified in Freund's complete adjuvant, and 300 pg were injected sub- cutaneously into the backs of female New Zealand White rabbits. Boosters of 300 pg of @ subunit emulsified in Freund's incomplete adjuvant were given a t 3, 6, and 12 weeks. One week after the final boost, blood was drawn by cardiac puncture.

Since attempts to extract n subunit from polyacrylamide gels were unsuccessful, we immunized rabbits directly with macerated polya- crylamide gel slices containing a subunit, as described by Bulinski and Borisy (8). The macerated gel was emulsified with Freund's complete adjuvant and injected subcutaneously into the backs of the rabbits. Boosters of approximately equal amounts of gel material were given at 2,4,8, and 12 weeks. Blood was drawn at 13 weeks by cardiac puncture. Anti-n antiserum was fractionated by chromatography on Protein A-Sepharose TM (Pharmacia).

In Vitro Translotion of RNA-Polysomal RNA was translated in a nuclease-treated rabbit reticulocyte lysate (9). Nuclease treatment was carried out exactly as described (9) except that 300 units/ml micrococcal nuclease was used. RNA was incubated in 2.5 mM CHRHgOH (10) for 5 min a t 29 "C and diluted 15-fold with reaction mixture. The final concentrations of the reaction components were as follows: 1.17 mg/ml polysomal RNA, 500 pg/ml yeast tRNA, 33 pg/ml Trasylol, 66% (v/v) nuclease-treated lysate, 25 mM Hepes/ KOH (pH 7.0), 100 mM potassium acetate, 1 mM magnesium acetate, 50 p~ all amino acids except methionine, 1 mM ATP, 0.2 mM GTP, 8 mM phosphocreatine, 0.5 mM spermidine, and 1.33 mCi/ml L-['%] methionine (>600 Ci/mmol). The translation reactions were incu- bated for 90 min a t 29 "C. Protein synthesis was estimated by tri- chloroacetic acid precipitation of reaction aliquots after hydrolysis of the aminoacyl-tRNA with 0.5 N NaOH.

Immunoprecipitation of Translotion Products-Aliquots (15-30 p l ) of the reaction mixture were adjusted to 70 p1 containing 1% SDS. Ten microliters of 10 mg/ml NEM were added, and the mixture was incubated for 10 min a t room temperature. Excess NEM was quenched by adding 4 p l of 200 mM dithiothreitol. (The NEM treat- ment was necessary for immunoprecipitation of pre-n subunit. For pre-fi subunit, this step was omitted and 10 pI of water substituted for the NEM.)

The SDS/NEM-treated mixture was then diluted to 800 pI by the addition of 250 pl of HzO, 400 pl of immunoprecipitation buffer (40 mM sodium phosphate (pH 7.5). 300 mM NaCI, 1% Triton X-100, 1% sodium deoxycholate, 4 mM EDTA, 20 mM methionine, and 10 mg/ ml bovine serum albumin), 25 p l of 5 mg/ml Trasylol, 50 pl of a 20% suspension of prewashed Pansorbin (Calbiochem-Behring), and 4 p1 of 0.5 M phenylmethylsulfonylfluoride in dioxane. The mixture was incubated for 15 min a t room temperature, and then the Pansorbin was sedimented in a Beckman microfuge for 2.5 min (11). Either 25 p1 of anti-n-IgG solution or 1 p1 of anti-fi antiserum was added to the supernatant, and the mixture was incubated for 30 min a t room temperature and then overnight a t 4 "C.

A volume of Pansorbin (20%, w/v) equal to twice the volume of antiserum was added to the immunoprecipitations, and the mixtures were incubated for 20-30 rnin a t room temperature. The mixture was layered over 0.55 ml of 0.5 M sucrose in wash buffer (20 mM sodium phosphate (pH 7.5), 150 mM NaCI, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 10 mM methionine, and 0.02% NaN3) and centrifuged for 4 min a t 13,000 rpm in a Sorvall HB-4 rotor. The supernatant was removed, the pellet was resuspended in 1 ml of wash buffer, and the cells were centrifuged for 1 min in a Beckman Microfuge B. This wash step was done twice more.

After the final wash, the supernatant was carefully removed, 80 pl of a solution containing 0.1 M triethylamine (pH 11.5) and 0.5% SDS were added (12), and the pellets were resuspended. After about 10 min of incubation a t room temperature, the samples were centrifuged

in the Microfuge for 3 min. The supernatant was added to 40 p l of electrophoresis sample buffer (4.1% SDS, 15% glycerol, 83 mM Tris- HCI (pH 6.8), 6.7% 2-mercaptoethanol, and 0.003% bromphenol blue), and 3 pl of 1 N HCI. After 15 min a t room temperature, the supernatant was applied to an SDS-polyacrylamide gel.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was per- formed using the discontinuous system of Laemmli (13), as described by Hokin et al. (14). After the run, the gel was prepared for fluorog- raphy using En3HanceTM (New England Nuclear) according to the manufacturer's directions. For fluorography, the gel was exposed to Kodak X-Omat AR-5 film a t -70 "C; a DuPont Cronex Lightning Plus screen was used.

For immunocompetition experiments, 5 pg of purified (Y subunit (3) or 2 pg of @ subunit (3) were added to the Pansorbin-treated supernatant before the antisera were added. Only 5 p l of anti-n or 0.5 p l of anti-fi was used in these experiments.

Purified f i subunit was labeled with ["C]formaldehyde (15) for use as a marker in SDS-PAGE. a subunit was labeled with '=I-labeled Bolton-Hunter reagent (3-(p-hydroxyphenyl)propionic acid succi- nimidyl ester) according to the protocol supplied by the manufacturer (New England Nuclear). Treatment with Bolton-Hunter reagent caused modifications in the cy subunits which resulted in diffuse bands after SDS-PAGE. This problem was reduced by incubation of the iodinated n subunit with 20 mM NEM for 15 min a t room temperature before dilution with electrophoresis sample buffer. This treatment increased the resolution of the al and n2 bands.

RESULTS

Structure of the N and /3 Precursors-Mature CY subunit from brine shrimp Na,K-ATPase appears as two bands on SDS-polyacrylamide gels (3); these bands are designated c y I

and a2 for the slower and faster migrating bands, respectively. The molecular weights for cy, and cy2 have been estimated to be 101,000 and 95,400, respectively (16). These two molecular forms have similar, if not identical, primary- and possibly higher-order structure (1,2) but differ in ouabain binding (4) and phosphorylation by [yR2P]ATP (5). The relative amounts of the two forms change during development of the brine shrimp, being approximately equal a t early times (-14 h) and changing to a 4:l N ~ / N ~ ratio at 48 h (1, 2). It is important to know whether a1 and a2 are produced by differential post- translational modification of a single precursor or result from the expression of two distinct precursors. The answer to this question would also suggest a mode of developmental control of the relative levels of c y I and cy2.

As seen from Fig. 1, the N subunit translation products are in fact two distinct proteins which migrate slightly slower than mature a1 and aZ. The estimated molecular weights of the two bands are 104,000 and 97,000, compared with apparent molecular weights of 99,000 and 92,000 for c y I and a*, respec- tively, in this gel system. We cannot yet say which translation product is the precursor to which mature form. The presence of two precursor forms of N subunit leads us to predict that the levels of a1 and a2 are controlled by differential expression of two messenger RNAs. Experiments are in progress to

1 2 FIG. 1. Immunoprecipitation of pre-a subunits. Translation

products from bound polysomal RNA were immunoprecipitated with anti-n-IgGs. The immunoprecipitate was submitted to SDS-PAGE and fluorographed. Lune 2 shows the immunoprecipitated pre-a sub- units. hrw I shows 12sII-labeled mature (I, and n2 subunits.

Site of Synthesis of Nu, K-A TPase Subunits 14219

determine whether this control is at the transcriptional or post-transcriptional level.

Whereas mature CY subunit appears as two discrete bands on polyacrylamide gels, mature /3 subunit appears as a diffuse region (Fig. 2). This diffuse @ band spans a molecular weight of 36,000 to 43,000 in this gel system. (Based on data using several gel systems, the true molecular weight of @ was esti- mated to be 44,000 (16).) Pre-@ subunit, however, appears as a single discrete band with a molecular weight of about 36,000. Like CY, @ is glycosylated but to a greater extent (3), so that difference in molecular weight between precursor and mature /3 is due a t least in part to the presence of oligosaccharide. The diffuse character of mature @ may be the result of microheterogeneity in glycosylation, such as has been seen in the electric eel @ subunit (17).

Identification of the Immunoprecipitated Translation Prod- ucts-To obtain further identification of the putative subunit precursors, we attempted to displace the binding of the trans- lation products to the anti-subunit antisera with mature sub- units. Fig. 3 shows these results for the putative pre-cu subunit. Mature CY subunit (lane 2) displaces the two peptides marked by the arrows, as well as some material in faint bands just below these two peptides. Neither mature p (lanes 3 and 4 ) nor phosphorylase a (lane 5) displaces the two peptides. We conclude that the two bands marked by arrows are a subunit precursors since they are displaced by mature a and they migrate near mature aI and a2 on SDS-polyacrylamide gels. We believe that the faint bands below the pre-a bands which are also displaced are proteolytic fragments since the a sub- unit is highly sensitive to proteases (18).

Fig. 4 shows the results of immunocompetition of pre-a by mature @. Purified /3 (lane 2) almost totally displaced pre-@ binding, while purified a (lane 3) did not displace any pre-@.

Site of Synthesis of the a and p Subunits-Figs. 5 and 6 provide evidence for the site of synthesis of the CY and p subunits. Lanes 1 and 2 of Fig. 5 show the translation products of bound and free polysomal RNA, respectively, immunopre-

1 2 3 4 5 FIG. 3. Immunocompetition of pre-a subunits. Lune I shows

translation products from bound polysomal RNA immunoprecipi- tated with anti-n-I&. Arrows indicate the positions of the pre-n subunits. Lane 2 is the same as lane I except that 5 pg of mature (Y

subunit were added to the immunoprecipitation reaction. Lunes 3-5 are the same as lane I except that 5 pg of mature 0 subunit (lane 3), 25 pg of mature @ ( l a n e 4 ) , or 25 pg of phosphorylase a ( l a n e 5) were added to the immunoprecipitation reaction.

+ 97400

f 66300

- 2 5 7 0 0

1 8 4 0 0

- 1 2 3

FIG. 4. Immunocompetition of pre-ff subunits. Lune I shows translation products from free polysomal RNA immunoprecipitated with anti$ antiserum. Lune 2 is the same as lane 1 except that 2 pg of purified 6 subunit were added to the immunoprecipitation reaction. Lune 3 is the same as lane I except that 20 pg of mature CY subunit were added to the immunoprecipitation reaction.

1 2 cipitated with anti-a antibody. Arrows point to the position of the pre-a subunits. Clearly, the a subunit precursors are

FIG. 2. Immunoprecipitation of Pre-ff subunit. Lune 1 shows present only in the translation products of membrane-bound “C-labeled mature 6 subunit. l ane 2 shows translation products from polysomal RNA. free polysomal RNA immunoprecipitated with anti+ antiserum and resolved by SDS-PAGE. Molecular-weight standards were phospho- Lanes and Of Fig. 6A show the products Of rylase b (95,400), bovine serum albumin (66,300), ovalbumin (42,800), bound and free PolYsomal RNA, respectively, immunoPreciP- wchymotrypsinogen (25,700), and &lactoglobulin (18,400). itated with anti-@ antiserum. In this case, pre-@ is primarily

14220 Site of Synthesis of Na,K-ATPase Subunits

”

1 2 FIG. 5. Site of synthesis of pre-a subunits. Lanes I and 2 show

translation products from bound and free polysomal RNA, respec- tively, immunoprecipitated with anti-a. Arrows point to the positions of the pre-n subunits.

A B

1 2 I 2 FIG. 6. Site of synthesis of pre-8 subunits. A, lanes I and 2

show translation products from bound and free polysomal RNA, respectively, immunoprecipitated with anti-13; B, same as A except that polysomes were prepared with 5 mM VRC rather than 10 mM.

in the translation products of free polysomal RNA. When polysomes were prepared in 5 mM VRC instead of 10 mM, the difference between the amount of pre-P in bound (Fig. 6B, lane I ) and free (Fig. 6B, lane 2) polysomal RNA became much more striking, further supporting the conclusion that pre-P is synthesized on free polysomes. The presence of pre- @ in the bound polysome fraction appears to be due to aggre- gation of polysomes induced by VRC during the preparation. More data supporting this assertion are presented below.

Aggregation of Polysomes Induced by VRC-In early exper- iments, we used 20 mM VRC and 0.5 mg/ml tRNA as ribo- nuclease inhibitors during polysome preparation. We noticed, however, that the amount of pre-P translation product syn- thesized from bound and free polysomal RNAs was nearly the same. This suggested that VRC was inducing aggregation of polysomes during their preparation.

We tested this hypothesis by preparing polysomes in 5, 10, and 20 mM VRC. (Lowering the concentration of VRC re- quired us to raise the level sf tRNA to 1 mg/ml and to use 1 mM EGTA (19) as an additional ribonuclease inhibitor.) In

A

1 2

B

I

C

2 1 2 FIG. 7. Polysome aggregation by VRC. Polysomes were pre-

pared in 5 mM ( A ) , 10 mM ( B ) , or 20 mM (C) VRC. Lanes 1 and 2 of each part show translation products from bound and free polysomal RNA, respectively, immunoprecipitated with anti$.

addition, we isolated polysomes from 16-h-old rather than 20- h-old nauplii since the younger nauplii have only about 1/4 as much ribonuclease as the older nauplii.’ Fig. 7 shows the distribution of pre-P translation product between bound (lane I ) and free (lane 2) polysomes prepared with 5 mM VRC (Fig. 7A) , 10 mM VRC (Fig. 7B) , and 20 mM VRC (Fig. 7 c ) . Clearly, the amount of pre-P in the bound polysome fraction, i.e. polysomes which pellet during the initial 5-min centrifugation a t 132,000 X g, increased with increasing VRC concentration.

DISCUSSION

The data we have presented show that the a subunit of the Na,K-ATPase in developing brine shrimp is synthesized on membrane-bound polysomes and that the B subunit is syn- thesized on free polysomes. The site of synthesis of a is consistent with the prediction of the signal hypothesis (20) that integral membrane proteins are synthesized on mem- brane-bound polysomes. To further clarify the mechanism of biogenesis of the a subunit, it will be necessary to determine whether insertion of the subunit occurs cotranslationally, as predicted by the signal hypothesis. The mechanism of inser- tion of the B subunit seems to be more complex. Since @ is synthesized on free polysomes, it might be expected that is inserted post-translationally, as are many organellar proteins (21-24) but not plasma membrane proteins. If post-transla- tional insertion does occur, it may occur independently of a or, alternatively, a may act as the membrane receptor (or “trigger” according to Wickner (25, 26)) for the insertion of /3 (see below). The latter model provides a simple mechanism for holoenzyme assembly, whereas the former model leaves the assembly question open.

Our data disagree with reports from Sabatini’s group con- cerning Na,K-ATPase biogenesis. In one report, Sabatini et al. (27) determined that a subunit was synthesized on free polysomes and that B subunit was synthesized on bound polysomes in cultured dog kidney (MDCK) cells. They sug- gested that p might act as the receptor for post-translational insertion of a. In a more recent abstract, Nabi et al. (28) reported that both a and B were synthesized on membrane- bound polysomes from rat brain. It is surprising that the findings in three systems should be different. An evolutionary difference in the mechanisms of synthesis and insertion seems unlikely since the structure of the Na,K-ATPase is well

* J. A. Fisher, L. A. Baxter-Lowe, and L. E. Hokin, unpublished observations.

Site of Synthesis of Na,K-ATPase Subunits 14221

conserved (29). In particular, rat brain and dog kidney a and p subunits react with the same anti-lamb kidney Na,K- ATPase antibody (29). We have seen immunological cross- reactivity between brine shrimp and electric eel Na,K- ATPase.3

A useful comparison may be made to the Ca2+-ATPase. Kyte (30) has proposed, based on certain structural similari- ties between the Na,K- and Ca2+-ATPases, that the two evolved from a common ancestor. He proposes that these, as well as the other active transport ATPases, share structural and functional features. If this is true, then it may be expected that their modes of synthesis are similar. (The Ca2+-ATPase has no subunit corresponding to p, so this similarity would apply only to cy.) In fact, it has been shown that the Ca2+- ATPase is synthesized on membrane-bound polysomes (31, 32) and cotranslationally inserted into membranes (33). Therefore if Kyte’s paradigm is correct, one would expect the synthesis of the a subunit of the Na,K-ATPase to occur on membrane-bound polysomes. Further studies on other sys- tems may help to clarify the seeming discrepancies.

We have also found that the a subunit is synthesized as two discrete precursor peptides, indicating that a1 and a2 are not produced by differential post-translational modification of a single translation product but are rather the translation products of two distinct messenger RNAs. In addition, the apparent molecular weights of the cell-free translation prod- ucts (104,000 and 97,000) suggest that at least one a subunit is synthesized as a precursor protein which is larger than the mature protein (99,000 and 92,000). Additional studies will be required to determine whether or not this is true for both a subunits.

The phenomenon of two a subunit forms may be fairly widespread. Two a subunit forms have also been observed in rat brain (34), canine brain (29), and chicken lens (35). Other investigators have been able to resolve two forms by heating canine kidney enzyme in the presence of SDS (36). In the abstract described above (28), Sabatini’s group also reports that there are two a subunit translation products in rat brain. Our data and Sabatini’s data suggest that the two forms in every case will be products of separate messengers.

The presence of separate a subunit translation products is likely to have implications for the mechanism of the devel- opmental regulation of a1/a2 content. One possibility is that the relative levels of messeger RNA for each LY form may change during naupliar development. Experiments are cur- rently underway to determine the changes in the relative levels of the a1 and a2 messengers during development by translating mRNA from nauplii of different ages. (As noted before, we cannot now state whether the higher-molecular- weight peptide is necessarily precursor to aI or the lower, precursor to a2.)

The funtional roles of the two a subunits are still unknown. In brine shrimp, a1 and ap may have a developmental role similar to other developmentally regulated isozymes. Swead- ner (34) provided evidence that the two forms found in brain are present in different cell types. Analysis of expression of the two forms by molecular biological techniques could be used to identify cells expressing one or the other form or to possibly identify repressed genes in cells not expressing one form. In any case, understanding the genetic regulation of the two forms should certainly aid in understanding the nature and function of their difference.

Finally, our data on the aggregation of polysomes by VRC

:’ d. A. Fisher and L. E. Hokin, unpublished observations.

should alert other workers to complications of its use. While we have successfully used it in our polysome preparations, it will not be possible to increase its concentration substantially for polysome preparation in tissues rich in ribonuclease. Using VRC in combination with other inhibitors may prove most effective for other investigators.

Acknowledgments-We wish to thank Constance Bowes for her technical assistance and Karen Wipperfurth for her assistance in preparing the manuscript.

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