Accumulation of alpha-mannosidase-1 in Dictyostelium discoideum requires many developmentally essential genes

alpha-Mannosidase-1, one of the earliest known developmentally controlled gene products in the cellular slime mold Dictyostelium discoideum, accumulates intracellularly during both axenic growth and development. The accumulation of alpha-mannosidase-1 activity prematurely ceases in all of 125 randomly isolated aggregation-deficient mutants at discrete times in development resulting in significantly reduced levels of cellular enzyme activity. This suggests that, unlike other developmentally controlled enzymes in this organism, the continued accumulation of alpha-mannosidase-1 activity is controlled by a large number of genes essential for early development. alpha-Mannosidase-1 misregulation and the aggregation-deficient phenotype are caused by the same mutation since (1) morphological revertants exhibit a coreversion to both fruiting ability and wild-type alpha-mannosidase-1 accumulation and (2) normal enzyme accumulation depends on the ability to aggregate and ultimately fruit in a conditional aggregation-deficient mutant. This type of regulation does not appear to be due to differences in enzyme secretion or changes in the overall rate of total protein synthesis. Aggregation-deficient mutants continue to synthesize protein beyond the time in development at which alpha-mannosidase-1 accumulation ceases. Our studies indicate that most of the 50-125 genes required for aggregation in Dictyostelium are also required for the normal accumulation of alpha-mannosidase-1 activity.     

α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

Lysosomal enzyme inactivation associated with defects in post-translational modification during development in Dictyostelium discoideum

The developmental accumulation of lysosomal alpha-mannosidase-1 activity in Dictyostelium discoideum is controlled at the level of de novo enzyme precursor biosynthesis. Aggregation-deficient mutants are defective with regard to the accumulation of alpha-mannosidase-1 activity beyond 8-16 h of development. We used enzyme-specific monoclonal antibodies to show that the activity defect in aggregation-deficient strains is not due to a lack of alpha-mannosidase-1-precursor synthesis or processing, or to preferential degradation of the mature enzyme protein. Instead, the defect is a result of enzyme inactivation: cells of aggregation-deficient strains contain significant amounts of inactive alpha-mannosidase-1 protein late in development. The alpha-mannosidase-1 inactivation phenotype is associated with a more general defect in lysosomal enzyme modification. A change in the post-translational modification system occurs during normal slime-mold development, as shown by differences in enzyme isoelectric point, antigenicity, and thermolability. We found that this change in modification does not occur in mutant strains blocked early in development. We propose a model in which pleiotropic mutations in early aggregation-essential genes can indirectly affect the accumulation of alpha-mannosidase-1 activity by preventing the expression of a developmentally controlled change in the post-translational modification system, a change which is required for the stability of several lysosomal enzymes late in development.     

Regulation of lysosomal α-mannosidase-1 synthesis during development in Dictyostelium discoideum

The cellular specific activity of lysosomal α-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. α-Mannosidase-1 is composed of two subunits (M_r = 58,000 and 60,000) which are derived from a common precursor polypeptide (M_r = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t_1/2 < 10 min). This indicates that the developmental accumulation of α-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional α-mannosidase-1 mRNA during development.     

A late developmental change in lysosomal enzyme sulfation specific to newly synthesized proteins in Dictyostelium discoideum

During development in Dictyostelium discoideum, several lysosomal glycosidases undergo changes in post-translational modification that are thought to involve differences in the extent of sulfation or phosphorylation, and appear to be required for the maintenance of cellular enzyme activity late in development. We have used monoclonal antibodies specific to the lysosomal enzyme alpha-mannosidase-1 to study the major late (12 hr) developmental change in the modification system. Pulse-chase experiments performed both early and late in development reveal that the substrate for the late form of modification is restricted to newly synthesized alpha-mannosidase-1 precursor protein. We have identified one modification difference between the two developmentally distinct isozymes of alpha-mannosidase-1: 35SO4 pulse-chase data show that the newly synthesized "late" enzyme precursor is significantly undersulfated in comparison with the enzyme synthesized early in development. This apparent lack of sulfation is associated with the lack of acquisition of endoglycosidase H resistance. By contrast, an aggregation-deficient mutant, which is defective with regard to the accumulation of alpha-mannosidase-1 activity late in development, synthesizes the "early" sulfated form of the enzyme throughout development. We conclude that the late developmental change in post-translational modification specifically involves one of the biochemical steps in which the N-linked oligosaccharide side chains of the newly synthesized alpha-mannosidase-1 precursor are modified by sulfation.     

A locus regulating N-acetylglucosaminidase synthesis during development in dictyostelium

The cellular specific activity of N-acetylglucosaminidase increases during development in Dictyostelium discoideum. A monoclonal antibody which specifically recognizes Mr 68,000 and 67,000 forms of N-acetylglucosaminidase was used to show that changes in the relative rate of enzyme synthesis during development parallel the pattern of enzyme accumulation. Developmental and regulatory mutants were isolated to study the relationship between development and enzyme accumulation. No evidence was obtained for any dependence of enzyme accumulation on those genes that are required for aggregation. However, a separate regulatory locus was identified which is involved in enzyme accumulation. Mutations in this gene, nagC, prevent enzyme accumulation during development by preventing an increase in the relative synthetic rate of N-acetylglucosaminidase. The accumulation of other enzymes is unaffected and the mutation causes no developmental defects other than those caused by the loss of N-acetylglucosaminidase activity. The nagC mutation, which is recessive, maps to linkage group VI and is therefore unlinked to the structural gene for N-acetylglucosaminidase.     

Glycosidases in cultured rat epididymal cells: enzyme activity, synthesis and secretion

During transit through the epididymis, spermatozoa acquire fertilizing the cell surface exhibits an altered glycoprotein pattern. Epididymal cells and their secretions contribute to these sperm-surface changes. To examine this process, epithelial cells from rat caput and cauda epididymidis were cultured and examined for the synthesis, processing and secretion of two glycoprotein-modifying enzymes, beta-galactosidase and beta-glucuronidase. Cells were cultured four days, incubated with D-2-[3H] mannose and L-[35S] methionine, and placed in isotope-free media. Levels of both cellular and secreted beta-galactosidase and beta-glucuronidase were determined by immunoprecipitation of cell homogenates or medium, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scintillation counting of bands. During a 1-h pulse, both caput and cauda cells synthesize two precursor forms of beta-galactosidase (Mr = 84,000 and 87,000), which are processed to the mature (Mr = 63,000) enzyme during a 24-h chase. Caput cells release a high molecular weight (HMW) form (Mr = 90-100,000) and mature beta-galactosidase into the media, but not the Mr = 84-87,000 precursor. On the other hand, cauda cells release mostly mature beta-galactosidase. Ratios of radiolabeled mannose/methionine demonstrate a 7-fold greater mannose content in the cellular precursor of beta-galactosidase than in total protein. Another glycosidase, beta-glucuronidase, is synthesized as a Mr = 78,000-precursor which is processed to the mature Mr = 72,000 form. Medium in which caput and cauda cells were cultured contains both mature enzyme and a Mr = 94,000 form, but no 78,000-precursor form. Ratios of radiolabeled mannose/methionine in the cellular precursor of beta-glucuronidase are 2-fold greater than ratios in the total glycoprotein. Secretion is the major pathway of turnover for several epididymal glycosidases, since more than 50% of the total is secreted/day. These results indicate that cultured epithelial cells from the epididymis synthesize glycosidases and that processing and release differ, depending on the enzyme and the epididymal segment from which the epithelial cells were isolated.     

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.     

Developmental regulation of rat lung Cu,Zn-superoxide dismutase.

In the present investigation we found that lung Cu,Zn-superoxide dismutase (SOD) activity (units/mg of DNA) increases steadily in the rat from birth to adulthood. The specific activity (units/micrograms of enzyme) of Cu,Zn-SOD was unchanged from birth to adulthood, excluding enzyme activation as a mechanism responsible for the increase in enzyme activity. Lung synthesis of Cu,Zn-SOD peaked at 1 day before birth and decreased thereafter to adult values. Calculations, based on rates of Cu,Zn-SOD synthesis and the tissue content of the enzyme, indicated that lung Cu,Zn-SOD activity increased during development owing to the rate of enzyme synthesis exceeding its rate of degradation by 5-10%. These calculations were supported by measurements of enzyme degradation in the neonatal (half-life, t1/2, = 12 h) and adult lung (t1/2 = greater than 100 h); the difference in half-life did not reflect the rates of overall protein degradation in the lung, since these rates were not different in lungs from neonatal and adult rats. We did not detect differences in the Mr or pI of Cu,Zn-SOD during development, but the susceptibility of the enzyme to inactivation by heat or copper chelation decreased with increasing age of the rats. We conclude that the progressive increase in activity of Cu,Zn-SOD is due to a rate of synthesis that exceeds degradation of the enzyme. The data also suggest that increased stabilization of enzyme conformation accounts for the greater half-life of the enzyme in lungs of adult compared with neonatal rats.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol

25-Hydroxycholesterol inhibits cholesterol biosynthesis by inhibiting the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Addition of 25-hydroxycholesterol to chicken myeloblasts caused a rapid inhibition of HMG-CoA reductase activity, producing approximately an 80% decrease in enzyme activity after 60 min. The mode of action of 25-hydroxycholesterol was determined by immunoprecipitating radiolabeled enzyme from 25-hydroxycholesterol-treated myeloblasts. The decline in enzyme activity due to addition of 25-hydroxycholesterol was not associated with increased levels of [32P]PO4 incorporation into the immunoprecipitated reductase polypeptide (Mr = 94,000). Hence, 25-hydroxycholesterol did not appear to regulate reductase activity by enzyme phosphorylation, as observed for other modulators of HMG-CoA reductase. However, 25-hydroxycholesterol was shown to inhibit reductase activity by causing a 350% increase in the relative rate of reductase degradation and a 72% decrease in the relative rate of reductase synthesis. These alterations in the rates of degradation and synthesis occurred rapidly (within 10-30 min after addition of 25-hydroxycholesterol) and can account completely for the 25-hydroxycholesterol-induced inhibition of enzyme activity. The rapid decline in the rate of synthesis of HMG-CoA reductase in 25-hydroxycholesterol-treated cells was not associated with concomitant changes in the levels of reductase mRNA; therefore, suggesting that 25-hydroxycholesterol must inhibit the rate of reductase synthesis by translational regulation. We also present evidence that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000.     

Turnover of succinyl-CoA:3-oxoacid CoA-transferase in glioma and neuroblastoma cells. Specific influence of acetoacetate in neuroblastoma cells.

The specific activity of succinyl-CoA:3-oxo-acid CoA-transferase (3-oxoacid CoA-transferase, EC 2.8.3.5) increases significantly during growth in culture in both mouse neuroblastoma N2a and rat glioma C6 cells. To investigate the mechanism(s) responsible for this, antibody specific for rat brain 3-oxoacid CoA-transferase was raised in rabbits. Immunotitrations of 3-oxoacid CoA-transferase from neuroblastoma and glioma cells on days 3 and 7 of growth after subculture showed that the ratio of 3-oxoacid CoA-transferase activity to immunoprecipitable enzyme protein remained constant, indicating that differences in specific activity of the enzyme at these times in both cell types reflect differences in concentration of enzyme protein. In glioma cells, the relative rate of 3-oxoacid CoA-transferase synthesis was about 0.04-0.05% throughout 9 days in culture. In contrast, the relative rate of synthesis of 3-oxo-acid CoA-transferase in neuroblastoma cells was about 0.07-0.08% on days 3, 5 and 7 after subculture, but fell to 0.052% on day 9. The degradation rates of total cellular protein (t1/2 = 28 h) and 3-oxoacid CoA-transferase (t1/2 = 46-50 h) were similar in both cell lines. The rise in specific activity of the enzyme in both cell lines from days 3 to 7 without a significant increase in the relative rate of synthesis reflects a slow approach to steady-state conditions for the enzyme secondary to its slow degradation. Differences in 3-oxoacid CoA-transferase specific activity between the two cell lines are apparently due to a difference of about 60% in relative rates of enzyme synthesis. The presence of 0.5 mM-acetoacetate in the medium significantly increased the specific activity of 3-oxoacid CoA-transferase in neuroblastoma cells during the early exponential growth phase. This treatment increased the relative rate of synthesis of 3-oxoacid CoA-transferase by 23% (P less than 0.025) in these cells on day 3, suggesting that substrate-mediated induction of enzyme synthesis is a mechanism of regulation of 3-oxoacid CoA-transferase.     

Biosynthesis of two lysosomal enzymes in macrophages. Evidence for a precursor of beta-galactosidase.

We have purified beta-galactosidase and beta-glucuronidase from macrophages of thioglycollate-treated mice using concanavalin A chromatography and immunoprecipitation. The apparent molecular weight of the beta-galactosidase subunit, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changed during a long term pulse-chase experiment. Following a 1-h pulse with [3H]leucine, radiolabel was present exclusively in an Mr = 82,000 form. However, after a 3-h chase in medium containing unlabeled leucine, most label migrated at Mr = 63,000, and at 24 h, all label was in the Mr = 63,000 form. Electrophoresis of peptides produced by cyanogen bromide cleavage of immunoprecipitates demonstrated structural similarities between precursor and mature forms. A mutation in the mouse, which is known to depress the rate of synthesis of beta-galactosidase in many cell types, proportionately decreased incorporation of [3H]leucine into both the Mr = 82,000 and 63,000 forms. Therefore, by kinetic, structural, and genetic evidence, the large molecular weight beta-galactosidase is a precursor of mature macrophage enzyme. No precursor of the Mr = 75,000 subunit of beta-glucuronidase was detected.     

Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii

A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo.     

Lipid Synthesis and Abundance of Acetyl CoA Carboxylase in Isochrysis galbana (Prymnesiophyceae) Following Nitrogen Starvation

Fatty acid content and the rate of lipid synthesis were measuredin the marine prymnesiophyte Isochrysis galbana grown undernitrogen starvation and in cultures recovering from nitrogendeprivation. Nitrogen starvation imposed a reduction in cellularsoluble protein content, variation in fatty acid compositionand reduction in the in vitro activity of the enzyme acetylCoA carboxylase. An increase in total fatty acid content isattributed to a differential reduction in cell division andthe rate of lipid synthesis. Recovery from nitrogen deprivationwas characterized by an increase in cellular soluble proteincontent and in the rate of lipid synthesis. Although the invitro activity of acetyl CoA carboxylase increased as the culturesrecovered from nitrogen starvation, the total cellular fattyacid content decreased, evidently due to an acceleration incell division. The relative cellular pool size of acetyl CoAcarboxylase determined by immunoblotting decreased under nitrogenstarvation conditions and increased as cells recovered fromit. Cellular accumulation of acetyl CoA carboxylase during recoveryfrom nitrogen starvation is ascribed to de novo synthesis ofthe enzyme that takes place in the cytoplasm. However, photosyntheticproteins such as ribulose bisphosphate carboxylase are synthesizedearlier than acetyl CoA carboxylase in the recovery process. (Received June 12, 1992; Accepted September 21, 1992)     

Induction of succinyl-coenzyme A:3-oxoacid coenzyme A-transferase during differentiation of 3T3-L1 cells

Differentiation of confluent 3T3-L1 preadipocytes to adipocytes in the presence of dexamethasone and 1-methyl-3-isobutylxanthine for 7 days resulted in a 4-fold increase in the incorporation of acetoacetate-carbon into fatty acids and in the activity of 3-oxoacid CoA-transferase, which catalyzes the first committed step in the conversion of acetoacetate to acetoacetyl-CoA. The increase in enzyme activity was due to an increase in the cellular content of the enzyme, as determined by immunoprecipitation of 3-oxoacid CoA-transferase from 3T3-L1 preadipocytes and adipocytes with rabbit antiserum specific for the rat brain enzyme. The 4-fold increase in enzyme activity was accompanied by a 2.7-fold increase in the average relative rate of synthesis of 3-oxoacid CoA-transferase (between Days 4 and 7). Additionally, the half-life of the enzyme increased 1.9-fold relative to the half-life of total protein, indicating that changes in both synthesis and degradation of 3-oxoacid CoA-transferase are responsible for alterations in its activity. Previous studies on the turnover of other enzymes that are induced during differentiation of 3T3-L1 cells have assigned changes in enzyme synthesis as the primary or sole mechanism for changes in enzyme activity. This report provides the first documentation that both enzyme synthesis and degradation play a role in regulating the enzyme activity of an enzyme during differentiation of 3T3-L1 cells.     

Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps

The precursor of the small subunit of ribulose bisphosphate carboxylase in Pisum sativum (relative molecular mass 20 000) is processed to the mature size (relative molecular mass 14 000) by the purified processing enzyme in two steps. The maturation proceeds via an intermediate of Mr 18 000. Both processing reactions may be carried out by the same enzyme although different residues are involved in the two cleavage sites. The second cleavage is inhibited if the precursor is pre-incubated with iodoacetate. The processing intermediate cannot be detected during the uptake of the precursor by intact isolated chloroplasts but iodoacetate-treated precursor is taken up and converted to a number of polypeptides of Mr 18 000 and below.     

Biosynthesis of the multiple forms of rabbit cardiac cathepsin D

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.     

Developmental regulation of the hepatocyte receptor for galactose-terminated glycoproteins

The receptor which recognizes glycoproteins that have had their terminal sialic acids removed, thus exposing penultimate galactose residues (asialoglycoproteins), was examined for expression in rat liver during development. The level of asialoglycoprotein receptor binding activity in fetal rat livers was present in very low amounts but rose dramatically at the time of birth and reached adult levels by the second day after birth. Using immunoquantitation methods, it was found that the increased binding capacity of rat liver for asialoglycoproteins during development reflected accumulation of receptor molecules rather than activation of previously existing ones. The relative rates of synthesis of the predominant polypeptide of Mr 42,000 and the lesser abundant polypeptides of Mr 50,000 and 58,000 which comprise asialoglycoprotein receptor were found to increase in livers of fetuses near term and attain adult synthesis rates around birth. Thus, the accumulation of receptor protein molecules during development reflected increased synthesis of receptor polypeptides. These results suggest that the different gene products which code for the three forms of the receptor are coordinately expressed during development. Copurifying with asialoglycoprotein receptor during ligand affinity chromatography were polypeptides of Mr 25,000 and 27,000. These polypeptides display several characteristics similar to hepatic mannose binding lectin described by others. Onset of synthesis of the mannose binding lectin during development was analogous to asialoglycoprotein receptor but, in contrast, did not reach adult synthesis rates immediately after birth.     

Sulfation and constitutive secretion of dopamine beta-hydroxylase from rat pheochromocytoma (PC12) cells

The biosynthesis and secretion of dopamine beta-hydroxylase were investigated by radiolabeling rat pheochromocytoma (PC12) cells in culture. Intracellular dopamine beta-hydroxylase from a crude chromaffin vesicle fraction and secreted dopamine beta-hydroxylase from culture medium were immunoprecipitated using antiserum made against purified bovine soluble dopamine beta-hydroxylase. Analysis of the immunoprecipitated enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that: 1) the membrane-bound form of the hydroxylase from crude secretory vesicle membrane extracts contained two nonidentical subunits in approximately stoichiometric amounts (Mr = 77,000 and 73,000); 2) the soluble hydroxylase from the lysate of these secretory vesicles was composed predominantly of a single subunit (Mr = 73,000); and 3) the hydroxylase secreted into the medium under resting conditions was also composed of a single subunit (approximate Mr = 73,000). All subunits of the multiple forms of hydroxylase were glycoproteins. Under resting conditions, the rate of secretion of hydroxylase was approximately 6% of total cellular enzyme/15 min. The secreted form of the hydroxylase incorporated [35S]sulfate, whereas no significant [35S]sulfate was incorporated into the cellular forms of enzyme. We propose that in addition to the dopamine beta-hydroxylase which is found in catecholamine storage vesicles and released during stimulus-coupled exocytosis, PC12 cells also have a constitutive secretory pathway for dopamine beta-hydroxylase and that the enzyme released by this second pathway is sulfated.