Mechanism of Biosynthesis of Soluble and Membrane-bound Forms of Dopamine @-Hydroxylase in PC 12 Pheochromocytoma Cells*

Dopamine &hydroxylase was present as 2 subunit forms (apparent M, = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [36S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The M, = 77,000 form was present in a crude membrane fraction, while the M, = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the M, = 77,000 and M, = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the M, = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the M, = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Trans- lation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine @-hy-droxylase is initially synthesized as a single polypep- tide (apparent M, = 67,000). The subcellular site of biosynthesis of dopamine &hydroxylase was deter- mined

tide (apparent M, = 67,000). The subcellular site of biosynthesis of dopamine &hydroxylase was determined by isolation of mRNA from free and membranebound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine &hydroxylase on membrane-bound polysomes.
These experiments suggest that both soluble and membrane-bound forms of dopamine &hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relatignship between the M, = 77,000 and the M. = 73,000 subunit forms of dopamine B-hydroxylase.
Dopamine 0-hydroxylase (EC 1.14.17.1) is the enzyme which catalyzes the formation of norepinephrine from dopamine (Kaufman and Friedman, 1965) and consequently it is the marker enzyme for the noradrenergic neuronal system (Geffen et al., 1969;Goldstein et al., 1972). In nerve terminals and in adrenal chromaffin cells, dopamine 0-hydroxylase is present in both the membrane and soluble content of the noradrenergic vesicles or chromaffin granules, respectively (Smith and Kirshner, 1967;Lagercrantz, 1976;Winkler, 1976). The soluble form of dopamine 0-hydroxylase can be secreted with the catecholamines (De Potter et al., 1969;Weinshilboum et al., 1971;Viveros et al., 1968). The soluble and membrane forms in the adrenal have been found to be similar immunochemically (Slater et al., 1981) and are reported to consist of four glycosylated subunits each with molecular weight of about 75,000 (Park et al., 1976;Fong et al., 1980;Rush and Geffen, 1980). Differences in the soluble and bound forms of dopamine Phydroxylase do appear, however, to exist. For instance, subtle differences in these forms were detected through peptide mapping (Slater et al., 1981). Charge-shift crossed immunoelectrophoresis has differentiated between an amphiphilic membrane-bound and a more hydrophilic soluble form (Bjerrum et al., 1979). These results have suggested that a small hydrophobic tail may anchor the enzyme in the membrane, and have raised the possibility of a biosynthetic relationship between the two forms. Recently, immature adrenomedullary vesicles in microsomal and Golgi fractions were shown to contain a high proportion of the amphiphilic form of dopamine @-hydroxylase (Helle and Serck-Hanssen, 1981). Pulsechase studies with perfused adrenals suggested that the kinetics of incorporation of soluble and membrane forms of dopamine P-hydroxylase into vesicles were similar, but different from that of newly synthesized chromogranin (Ledbetter et al., 1978). On the other hand, Winkler et al. (1972) showed in labeling experiments with radioactive amino acids that the membrane proteins of adrenal-medullary granules were labeled considerably later than proteins in the soluble content. The membrane and releasable form of dopamine @-hydroxylase appeared to turn over at different rates, and it was suggested that the two forms are synthesized as different molecules and that there is no appreciable exchange between the membrane-bound and soluble pools of dopamine 0-hydroxylase (Gagnon et al., 1976;Winkler, 1977).
We have utilized the PC12 pheochromocytoma cell line to investigate the mechanisms of biosynthesis of the soluble and membrane-bound forms of dopamine @-hydroxylase. Established from a transplantable rat pheochromocytoma, PC12 cells proliferate in serum-containing medium and possess the differentiated properties of chromaffin cells, including the presence of chromaffin granules, and the synthesis, storage, and release of dopamine and noradrenaline Tischler, 1976, 1982;Greene and Rein, 1977). These cells have the advantage of providing large amounts of homogeneous material for biochemical analysis.
In this paper, we report that in PC12 cells, subunits of dopamine @-hydroxylase isolated by immunoprecipitation, are present in soluble and membrane forms (apparent M , = 73,000 Mechanism of Biosynthesis and 77,000, respectively). Both subunit forms are glycosylated by the tunicamycin-sensitive pathway. Evidence is presented to support a precursor-product relationship between these forms.
Immunoprecipitation-The cells were washed twice with 0.15 M NaCI, 10 mM Na phosphate, pH 7.2 (PBS'), then scraped with a rubber policeman into PBS containing 2% sodium dodecyl sulfate. The cell lysate was sonicated (Kontes, micro-ultrasonic cell disrupter), and if not processed immediately, was stored at -20 "C. For immunoprecipitations, the cell lysates were boiled 2 min, cooled, diluted with 4 volumes of solution A (0.19 M NaCI, 50 mM Tris-HCI, pH 7.4, 5 mM EDTA, 2.5% Triton X-100, 100 units/ml of Trasylol), and processed for immunoprecipitation by the indirect procedure with protein A Sepharose (Goldman and Blobel, 1978) as previously described (Sabban et al., 1981). An excess of specific antiserum (usually 3 p1 undiluted) or preimmune serum was used. In some experiments, the nonspecific background of the immunoprecipitates was decreased by first incubating with preimmune serum, removing insoluble material at 12,800 X g for 30 min, and processing of the supernatant for immunoprecipitation with anti-dopamine 0-hydroxylase antiserum. After washing the protein A-Sepharose five times with solution A containing 0.1% SDS, the immunoprecipitated proteins bound to protein A-Sepharose were dissolved by boiling 2 min in 60 PI of 50 mM Tris-phosphate, pH 6.7, containing 1 M dithiothreitol, 10% sucrose, 10% SDS, 20 mM EDTA. The beads were removed by centrifugation at 12,800 X g for 3 min and the supernatant was analyzed by gel electrophoresis.
Gel Electrophoresis-Gel electrophoresis was carried out in 6-12% polyacrylamide slab gels in the presence of SDS according to a modification (Kreibich and Sabatini, 1974) of the procedure ofMaize1 (1971). The distribution of the radioactive proteins relative to I4Clabeled molecular weight markers was determined by fluorography with sodium salicylate (Chamberlain, 1979) and exposure to prefogged (Laskey and Mills, 1975)  of Dopamine @-Hydroxylase 7813 disrupter) in ice-cold lysis solution (5 mM Tris-HC1, containing 100 pg/ml phenylmethylsulfonyl fluoride and 200 units/ml of Trasylol). Nuclei and unlysed cells were removed by centrifugation at 5,000 X g for 10 min and the postnuclear supernatant was centrifuged at 100,000 x g (Ti-40.3 rotor, Beckman Instruments) for 60 min at 4 "C. The supernatant was collected and the pellet was washed twice and suspended by vigorous vortexing into "lysis solution" containing either 0.1% Triton X-100 (when assayed for enzyme activity or for determination of catecholamines) or 2% sodium dodecyl sulfate (when processed for immunoprecipitation).
Assays-The activity of dopamine @-hydroxylase was assayed according to Nagatsu and Udenfriend (1972), except that bovine serum albumin and 0.1% Triton X-100 was added to each of the samples. Freshly prepared cell lysates or soluble and membrane fractions of cell lysates were used. Protein was determined according to Bradford (1976) with the Bio-Rad assay kit. The catecholamines were extracted with alumina and analyzed by high pressure liquid chromatography as previously described (Rabey et al., 1981).
Preparation of mRNA-The RNA from rat adrenals or bovine adrenal medulla was prepared according to Liu et al. (1979). Free and membrane-bound polysomes were prepared from bovine adrenal medulla according to the procedure developed by Ramsey and Steele (1976, 1977, 1979 for rat liver polysomes. The RNA in the polysome pellet was extracted with guanidine hydrochloride by a modification of the procedure of Cox (1964), as previously described (Sabban et al., 1981). The poly(A) mRNA was purified by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972) and used for translation experiments.
Cell Free Translations and Immunoprecipitation-In vitro translation was carried out in a wheat germ lysate at 25 "C (Roman et al., 1976;Sabban et al., 1982) with [35S]methionine. For immunoprecipitation, we used equal cpm of [35S]methionine-labeled protein, as determined by trichloroacetic acid precipitation under conditions which cleave aminoacyl-tRNA (Mans and Novelli, 1964). The translation solution was adjusted to 2% SDS, boiled 2 min, and processed for immunoprecipitation as described above. Homologous antiserum (guinea pig antiserum to rat dopamine @-hydroxylase or goat antiserum to bovine dopamine @-hydroxylase) was used with translations of rat or bovine mRNA, respectively.

Subunit Forms of Dopamine @-Hydroxylase in PC12 Cells-
In order to characterize the subunit forms of dopamine phydroxylase, PC12 cells were labeled with ["Slmethionine for several hours. The cell lysate was immunoprecipitated with antibodies prepared against rat dopamine @-hydroxylase, the immunoprecipitates were analyzed on 6-12% linear gradient polyacrylamide slab gels in SDS, and the radioactive proteins were detected by fluorography. Two specific subunit forms of immunoprecipitated dopamine P-hydroxylase were consistently detected with apparent molecular weights of 73,000 and 77,000 (Fig. 1). These forms are present in approximately equal amounts as indicated by their approximately equal intensity of ["S]methionine label. When cells were labeled for longer periods of time (24 h), these two bands were still present although they were somewhat more diffuse.
T h e presence of ascorbic acid, which has been found to stabilize dopamine 0-hydroxylase against proteolysis in vitro (Wong et al., 1981), had no effect on the distribution of the subunit forms of dopamine @-hydroxylase. Thus, when 1 and 5 mM ascorbic acid was freshly added to PC12 cells during a 2-h preincubation and a 2-h labeling period, the two forms were still present in near equal amounts.
Subunit Forms in Soluble and Membrane Fractions-Dopamine P-hydroxylase in the adrenal is known to exist in membrane and soluble fractions of chromaffin granules. We carried out a crude subcellular fractionation of [35S]methionine-labeled PC12 cells to find the localization of the subunit forms of dopamine @-hydroxylase. Cells were lysed by brief sonication in hypotonic solution in the presence of protease inhibitors. A crude membrane fraction was prepared. with antisera specific for dopamine Ij-hydroxylase (lane H) or with preimmune sera (lane> C), analyzed on a B-125; polyacrylamide slab gel in the presence of SDS and processed for fluorography as described under "Experimental Procedures" and compared to "C-labeled molecular weight standards. The total protein profile in lane A (130,000 trichloroacetic acidprecipitable cpm) was exposed to film for 8 h while lanes H and (' were immunoprecipitated from 1.5 x 10" trichloroacetic acid-precipitahle cpm and exposed to film for 4 days. Similar results were obtained in over %O experiments.
dodecyl sulfate and processed for immunoprecipitation with anti-dopamine /j-hydroxylase antiserum.
The 77,000-M, subunit form was greatly enriched in the membrane fraction, while the 78,000-M, subunit form was greatly enriched in the soluble fraction (Fig. 2). Analysis of the fractions for catecholamines indicated that the norepinephrine and dopamine were soluble and hence, that under these conditions, the storage sites were lysed.
The assay of dopamine /j-hydroxylase activity was carried out on the membrane and soluble fractions (Table I). Dopamine @-hydroxylase activity was recovered in both fractions, supporting the view that both subunit forms are indeed active dopamine /j-hydroxylase.
However, under the conditions used, the membrane form has an almost lo-fold higher specific activity when expressed per mg of total protein. Since the similar labeling of the 73,000-and 77,000-M, subunit forms with [""Slmethionine suggests that they are present in near equal molar amounts, and since both fractions contained similar amounts of protein (Table I), the variation in activity may indicate that the 77,000-M, membrane form is intrinsically more active. Alternatively, it may be less susceptible to inactivation during the fractionation. Since phosphorylation is known to modulate the activity of many enzymes including tyrosine hydroxylase (Raese et al., 1977;Latendre et al., 1977;Joh et al., 1978;Markey et al., 1980;Yamauchi and Fujisawa, 1979;Lazar et al., 1982), we examined whether the two subunit forms might be differentially phosphorylated.
PC12 cells were labeled for 2 h with ["'Plorthophosphate, and dopamine /j-hydroxylase was immunoprecipitated from the resulting cell homogenates and resolved by SDS-polyacrylamide gel electrophoresis. Radioau-tography of the gels revealed no detectable phosphorylation of either of the two subunit forms of dopamine [Ghydroxylase, even though phosphorylation of many other proteins was readily detectable.
(&xJsykxtion of I)opamine /j-Hydroxylase-Dopamine fihydroxylase is known to be a glycoprotein (Wallace et al., 1973;Ljones et al., 1976;Geissler et al., 1977). It has recently been shown that soluble and membrane-bound dopamine @hydroxylase in bovine adrenal chromaffin granules have an indistinguishable sugar composition (Fischer-Colbrie et al., 1982). Thus, the PC12 cells were labeled with radioactive The supernatant and membrane fractions were obtained from a postnuclear supernatant of PC12 cells which had been labeled with [""S]Met for 4 h as described under "Experimental Procedures." The soluble fraction contained 52% and the memhrane had 48'; of the trichloroacetic acid-precipitable [%I Met label. The solutions were adjusted to contain 2% sodium dodecyl sulfate and aliquots (5 ~1) were taken to analyze the total profiles of proteins in the soluble (Innc A), and membrane fractions (Lance c'), and to compare these to the postnuclear supernatant (lone H). The remainder of the material in each fraction (145 ~1) was immunoprecipitated with specific antibodies against dopamine $-hydroxylase. The immunoprecipitates from soluble, membrane-bound, and total postnuclear supernatant are shown in /anc~.s 11, E', and E:', respectively. This experiment was repeated twice and comparable results were obtained.  68K-sugars and then processed for immunoprecipitation to detect whether both subunit forms are glycosylated. After treatment for 4 h with 13H]mannose, both subunit forms of dopamine fi-hydroxylase were labeled, suggesting that both are glycosylated (Fig. 3). In longer labeling (several days), the immunoprecipitated material was extremely diffuse with electrophoretic mobility corresponding to M, = 73,000-77,000. Labeling with ['Hlfucose also revealed two subunit forms which incorporate similar amounts of ["Hlfucose (not shown).
To further delineate the glycosylation of the multiple forms of dopamine P-hydroxylase, PC12 cells were treated with tunicamycin.
The latter drug blocks glycosylation by interfering with the formation of dolichol-bound N-acetyl glucosamine derivatives (Struck and Lennarz, 1977;Tkacz and Lampen, 1975  should be noted that, while similar numbers of trichloroacetic acid-precipitable counts were used for each immunoprecipitation shown in Fig. 4, the immunoreactive forms in the tunicamycin-treated cells were considerably reduced, suggesting that the nonglycosylated forms may be more rapidly degraded. Quantification of densitometer scans of the results, shown in Fig. 4, showed that in tunicamycin-treated cells the relative amount of immunoreactive dopamine P-hydroxylase in the two new specific polypeptides is 16% of the original forms. These results also showed that the antibodies recog- . Approximately equal amounts of cells (4 X lo6 cells) corresponding to 2 x lo6 cpm (control), 1.7 X lo6 cpm (6 pg/ml of tunicamycin), or 1.6 X lo6 cpm (10 pm/ml of tunicamycin) of [3LS]Met-labeled protein was used for the immunoprecipitations.
Arrours indicate positions of bands that were specifically immunoprecipitated by the antiserum to dopamine fi-hydroxylase. tides immunoprecipitated, thus ruling out the possibility that the nonglycosylated material was not as efficiently immunoprecipitated under our experimental conditions.
Relationship between the Subunit Forms-Pulse-chase experiments were carried out to determine if there is a biosynthetic relationship between the multiple forms. When PC12 cells were labeled with ["sSS]methionine for 5 min, only the 77,000-M, subunit form was detected (Fig. 5). A chase with unlabeled methionine subsequently showed the appearance of the 73,000-M, subunit form in near equal amounts to the 77,000-M, subunit form by 90 min. This proportion between the 73,000-and 77,000-M, subunit forms remained constant for at least several hours (Fig. 5). This is consistent with results in nerve growth factor-treated PC12 cells (Sabban et al., 1983), in which the 73,000-M, subunit predominates, and in which pulse-chase experiments show that the 77,000-M, subunit form is synthesized first. Due to the large reduction in the amount of dopamine &hydroxylase in tunicamycintreated cells, it was not feasible to carry out pulse-chase experiments with them, and to directly determine whether the 67,000-M, form is a precursor for the 63,000-M, form observed in tunicamycin-treated cells.
Translation Product for Dopamine P-Hydroxylase-In order to confirm that only one form of dopamine /?-hydroxylase is initially synthesized, we examined the translation product for dopamine &hydroxylase in a cell-free system. Isolation of newly synthesized dopamine @-hydroxylase, using mRNA from PC12 cells, gave a high nonspecific background, probably since low molecular weight mRNA's are more efficiently translated. Therefore, we examined the translation product for dopamine P-hydroxylase using mRNA from total rat adrenals, or from bovine adrenal medullae, both of which contain higher concentrations of dopamine &hydroxylase (and presumably the corresponding mRNA) than the PC12 cells.
The mRNA was translated in a cell-free system and the newly synthesized polypeptide of dopamine @-hydroxylase isolated by immunoprecipitation. With rat adrenal mRNA, only one specific polypeptide (apparent M, = 67,000) was observed in the higher molecular weight region (not shown). Its electrophoretic mobility was identical to that of the larger form of dopamine @-hydroxylase in tunicamycin-treated PC12 cells. In translation of mRNA from bovine adrenal medulla (Fig. 6), a single polypeptide (apparent M, = 67,000) is obtained, along with an additional band of apparent M, = 32,000; the latter had an identical electrophoretic mobility to newly synthesized phenylethanolamine N-methyltransferase (Sabban et al., 1982). These results, although obtained in adrenals, support the findings in PC12 cells that dopamine P-hydrox- ylase is initially synthesized as a single polypeptide (apparent M, = 67,000).
Site of Synthesis of Dopamine /?-Hydroxylase-The previously mentioned results with tunicamycin suggested that dopamine @-hydroxylase may be synthesized on membranebound polysomes since tunicamycin interferes with core glycosylation which takes place in the endoplasmic reticulum. The oligosaccharide-lipid donor for glycosylation of asparagine residues has been found localized on the luminal side of microsomes (Snider and Robbins, 1982).
In order to ascertain the subcellular site of synthesis of dopamine P-hydroxylase, free and membrane-bound polysomes were prepared from bovine adrenal medulla. The mRNA was extracted, and used in cell-free translations in a wheat germ extract system. This procedure yielded 2.6 times more free than bound mRNA. Both were active in directing protein synthesis. Immunoprecipitation of equal amounts of trichloroacetic acid-precipitable translation products with antibodies to dopamine P-hydroxylase localized the mRNA coding for dopamine &hydroxylase exclusively on membranebound polysomes (Fig. 6). The mRNA for the 32,000-Mr polypeptide, which was precipitated with antibodies to dopamine &hydroxylase, was predominately on free polysomes, while the single polypeptide (Mr = 67,000) was obtained only in immunoprecipitates of translation products from membrane-bound polysomes.

DISCUSSION
Subunit Forms of Dopamine 0-Hydroxylase-The PC12 cells have been shown to synthesize two subunit forms of dopamine /3-hydroxylase with apparent M, = 73,000 and 77,000. These forms were separated electrophoretically on gradient polyacrylamide-SDS slab gels of immunoprecipitated dopamine @-hydroxylase. Two forms of dopamine p-hydroxylase were detected previously by charge shift immunoelectrophoresis of dopamine @-hydroxylase from adrenal medulla (Bjerrun et al., 1979). However, separation of two subunit forms, to our knowledge, has not previously been described on polyacrylamide gels, although dopamine @-hydroxylase often is represented as a rather diffuse band. The differentiation between these two forms probably reflects the enhanced sensitivity of long gradient polyacrylamide slab gels over the commonly used 7.5 or 10% polyacrylamide gels as well as the greater resolution of fluorography over sliced gels and scintillation counting. It should, however, be noted that in longer labeling times these bands appeared more diffuse and the distinction was somewhat less clear-cut. This may reflect alterations in the carbohydrate moiety during the lifetime of the molecule.
The membrane form appeared to have a 10-fold higher specific activity than soluble dopamine @-hydroxylase in this system. However, the possibility that the soluble form may be inactivated more rapidly cannot be ruled out. In studies on dopamine @-hydroxylase in the rat adrenal, Ciaranello et al. (1975) also reported a much higher (6-fold) specific activity of dopamine @-hydroxylase in a particulate than a soluble fraction obtained from a total homogenate. They showed by immunotitration that the differences were not due to altered amounts of enzyme, but rather that the particulate fraction has a higher homospecific activity (activity/amount of the specific protein measured immunologically (Rush et at., 1974)). In contrast, others (Rush et al., 1974;Helle and Serck-Hanssen, 1981) who studied the specific activity of dopamine P-hydroxylase in isolated granules found homospecific activity of the soluble fraction about 5-fold higher than membrane-bound forms of dopamine @-hydroxylase.
While immunological data are subject to the possibility of detecting a contaminating protein, this is unlikely in the present study. The presence of enzyme activities in the soluble and membrane-bound fractions would indicate that both subunit forms (M, = 73,000 and 77,000) indeed represent dopamine @-hydroxylase. However, the order of magnitude difference in specific activity between them makes the argument ambiguous, since small contamination of the soluble form with the membrane form could alter the results. More conclusive evidence that both forms probably are indeed bona fide dopamine @-hydroxylase comes from the pulse-chase experiments, particularly in nerve growth factor-treated cells in which the 77,000-M, form is almost completely converted to the 73,000-Mr form (Sabban et al., 1983).
The pulse-chase experiment on untreated PC12 cells showed that initially the 77,000-Mr subunit form is synthesized and apparently processed to the 73,000-Mr form in 15-90 min. The similarity of the labeling pattern between 90 min to 4 h indicates that equilibrium has been achieved and that the label does represent total protein, and not just rate of synthesis. It should, however, be noted that during the chase (Fig. 5), there is an increase in the amount of both subunits. We did not detect any immunoreactive higher molecular weight precursor. The increase in radioactivity probably reflects variation in the "actual" labeling time during the chase, assuming a lag for entry of [35S]methionine into the cellular pool and a similar lag for entry of the unlabeled chase methionine. Moreover, any nascent chains whose biosynthesis began during the pulse, and which are completed during the chase, would show up in the immunoprecipitate. In support of these possibilities, a comparable experiment employing antiserum to tyrosine hydroxylase also showed increased labeling of tyrosine hydroxylase during the chase period, although in this case there is no processing.
Site of Synthesis of Dopamine P-Hydroxylase-We have shown directly that dopamine @-hydroxylase is synthesized exclusively on membrane-bound polysomes of bovine adrenals. While parallel experiments could not be carried out with PC12 cells, it appears likely that a similar mode of synthesis pertains in this system as well. Such a mechanism is consistent with the site of synthesis of a protein destined for secretion (as occurs in a variety of systems) and of most plasma membrane proteins (Palade, 1975;Blobel, 1978;Sabatini and Kreibich, 1976;Sabatini et al., 1982). Thus, dopamine @-hydroxylase would be expected to contain an NH2-terminal signal sequence which directs vectorial discharge into the endoplasmic reticulum and which is subsequently removed (Blobel and Sabatini, 1971;Blobel and Dobberstein, 1975). However, surprisingly the electrophoretic mobility of the translation products is identical to one of the forms in tunicamycintreated cells. Perhaps, dopamine @-hydroxylase may contain an internal insertion signal similar to ovalbumin (Lingappa et al., 1979) or the major transmembrane erythrocyte glycoprotein, band 3 (Sabban et al., 1981;Braell and Lodish, 1982). The mechanism by which dopamine P-hydroxylase is inserted into membranes should be investigated further.
The exclusive localization of mRNA for dopamine @-hydroxylase on membrane-bound polysomes would indicate, as previously suggested (Gagnon et at., 1976), that the portion of the dopamine @-hydroxylase which is detected in the supernatant during subcellular fractionation is due to leakage from vesicles during the preparation and that dopamine (3hydroxylase is not present in a free form in the cytosol. There appears to be only one translation product for dopamine @-hydroxylase, with apparent M, = 67,000. This value may seem somewhat low since subunits of bovine dopamine @-hydroxylase, with electrophoretic mobility corresponding to apparent molecular weight of 73,000-75,000, is reported to contain about 5% carbohydrate (Wallace et al., 1973;Geissler et al., 1977;Fischer-Colbrie et al., 1982). However, it is not unusual for glycoproteins, and particularly sialoglycoproteins to have somewhat anomalous electrophoretic mobilities. It should be noted that immunoprecipitation from the translation with anti-dopamine @-hydroxylase antisera also isolates a polypeptide with identical electrophoretic mobility to newly synthesized phenylethanolamine-N-methyltransferase (apparent M, = 32,000). Moreover, this polypeptide is much more prominent in immunoprecipitates from free polysomes, and thus, is unlikely to represent a degradation product of dopamine P-hydroxylase. These results are interesting in light of recent suggestions by Joh and co-workers of possible similarities in domains between the enzymes involved in the synthesis of catecholamines (Joh et al., 1981).
The results presented here, as well as those by Helle and Serck-Hanssen (1981), suggest post-translational processing of dopamine @-hydroxylase from the membrane to soluble form. We cannot, howevererule out the possibility of formation of the 73,000-Mr and 77,000-Mr forms at different rates from a higher molecular weight precursor which is unrecognized by antiserum to native dopamine @-hydroxylase. However, the processing of the subunit form to the 73,000-Mr form seems the more likely interpretation of the data. This event occurs relatively quickly (within about 15-90 min), and it appears from the pulse-chase data that, if processing does not occur initially, the distribution of the two forms remains relatively intact. Glycosylation does not appear to be necessary for this process, since in tunicamycin-treated cells, two subunit forms (albeit of lower apparent molecular weight) are obtained. These results indicate that the nonglycosylated forms may be less stable since they are reduced by about 6fold in the tunicamycin-treated cells. Indeed, it has been suggested for a number of proteins that the function of the carbohydrate moiety is to prevent degradation, and there is evidence that the nonglycosylated forms of several proteins, such as fibronectin or the acetylcholine receptor, are more susceptible to proteolysis (Olden et al., 1982). In summation, the experiments presented here lead to the following model for the biosynthesis of dopamine p-hydroxylase. The enzyme is synthesized in the endoplasmic reticulum on membrane-bound polysomes (Mr = 67,000) and rapidly glycosylated to a 77,000-Mr form. This membrane-bound form can be processed to a 73,000-Mr soluble form relatively rapidly within 15-90 min. If not converted then, the distribution remains relatively constant, at least for several hours.
These findings on the mechanism of biosynthesis of dopamine &hydroxylase should be helpful in designing further experiments on the biogenesis of chromaffin granules and neuronal vesicles. In particular, it would be of considerable interest to elucidate the factors which regulate whether do-