Hepatic lipase. Synthesis, processing, and secretion by isolated rat hepatocytes

Hepatic lipase, a glycoprotein synthesized and secreted by the hepatocyte, binds to sinusoidal endothelium where it is involved in metabolism of lipoprotein phospholipid and triglyceride. To better understand the regulation of hepatic lipase, we investigated the synthesis, post-translational processing, and secretion of the enzyme by isolated rat hepatocytes. Metabolically labeled [35S]methionine hepatic lipase protein, produced by the collagenase-dispersed hepatocytes, was immunoisolated from detergent-solubilized cells and incubation medium at designated times, using a polyclonal rabbit anti-rat hepatic lipase antibody raised against hepatic lipase purified to homogeneity from rat liver post-heparin perfusates. Following polyacrylamide gel electrophoresis and fluorography, radiolabeled hepatic lipase was quantitated by densitometry. Newly synthesized hepatic lipase was rapidly secreted and accumulated in the medium as a 59,000-dalton protein in a manner consistent with a constitutive process. An intracellular 53,000-dalton precursor of the mature 59,000-dalton hepatic lipase was identified by immunoprecipitation. The 53,000-dalton form could also be generated by endoglycosidase digestion of the secreted 59,000-dalton protein. In pulse-chase experiments, the 53,000-dalton protein was converted into the 59,000-dalton form. A 47,000-dalton form of hepatic lipase was immunoisolated from cell lysates only after tunicamycin treatment and could be generated from the secreted 59,000-dalton enzyme by prolonged endoglycosidase digestion. These data show that hepatic lipase is synthesized and rapidly secreted by isolated rat hepatocytes. Further, an intracellular 47,000-dalton precursor peptide can be identified after tunicamycin treatment, which may represent the hepatic lipase polypeptide, presumably after removal of its signal sequence; a 53,000-dalton partially glycosylated peptide exists as a major precursor form in the cell; and the mature 59,000-dalton hepatic lipase is present in the hepatocyte, but it is rapidly secreted.     

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.     

Biosynthesis and turnover of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus

Antiserum was raised in rabbits against a bile canalicular glycoprotein of Mr = 110,000 purified to homogeneity from of rat liver. The antisera specifically immunoprecipitated a Mr = 110,000 polypeptide from hepatocytes metabolically labeled with [35S]methionine. When hepatocytes in primary culture were incubated with tunicamycin before labeling with [35S]methionine in the presence of tunicamycin, the major polypeptide immunoprecipitated by the specific antiserum from Triton X-100 extracts of cells had a molecular weight of 59,000. Enzymatic removal of N-linked carbohydrates from the Mr = 110,000 glycoprotein by N-glycanase digestion also yielded a polypeptide with minimum Mr = 59,000. In pulse-chase experiments using [35S]methionine, the Mr = 110,000 protein detected by the specific antisera first appears as Mr = 85,000 and 75,000 intermediate species which are endoglycosidase H sensitive. The Mr = 85,000 intermediate form is lost first with time followed by the Mr = 75,000 form giving rise to the Mr = 110,000 form that is endoglycosidase H resistant. Neuraminidase digestion of the Mr = 110,000 form generated an Mr 85,000 form but with a different carbohydrate structure than the intermediate Mr 85,000 form detected in the pulse-chase experiments. The time required to accomplish the processing of the Mr = 85,000 and 75,000 forms is relatively slow. Finally, the terminal sugars are added and the mature Mr = 110,000 glycoprotein is rapidly transported to the cell surface. A minimum time of 90 min is required for the Mr = 110,000 bile canalicular glycoprotein to be synthesized, processed, and reach the cell surface which is long relative to the time required (10 min) for another domain-specific protein, the receptor for asialoglycoproteins, to reach the sinusoidal surface. The Mr = 110,000 bile canalicular glycoprotein turns over in the bile canalicular domain with a half-life of 43 h while the asialoglycoprotein receptor turns over in the sinusoidal domain with a half-life of 23 h.     

Biosynthesis and processing of lysosomal cathepsin L in primary cultures of rat hepatocytes

The biosynthesis and proteolytic processing of lysosomal cathepsin L was studied using in vitro translation system and in vivo pulse-chase analysis with [35S]methionine and [32P]phosphate in primary cultures of rat hepatocytes. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of a primary translation product of an immunoprecipitable 37.5-kDa cathepsin L in vitro. The 37.5-kDa form was converted to the 39-kDa form when translated in the presence of dog pancreas microsomes. During pulse-chase experiments with [35S]methionine in cultured rat hepatocytes, cathepsin L was first synthesized as a 39-kDa protein, presumably the proform, after a short time of labeling, and was subsequently processed into the mature forms of 30 and 25 kDa in the cell. On the other hand, considerable amounts of the proenzyme were found to be secreted into the culture medium without further proteolytic processing during the chase. The precursor and mature enzymes were N-glycosylated with high-mannose-type oligosaccharides, and the proenzyme molecule contained phosphorylated oligosaccharides. The effects of tunicamycin and chloroquine were also investigated. In the presence of tunicamycin, a 36-kDa unglycosylated polypeptide appeared in the cell and this protein was exclusively secreted from the cells without undergoing proteolytic processing. These results suggest that cathepsin L is initially synthesized on membrane-bound polysomes as a 37.5-kDa prepropeptide and that the cotranslational cleavage of the 1.5-kDa signal peptide and the core glycosylation convert the precursor to the 39-kDa proform, which is subsequently processed to the mature form during biosynthesis. Thus, the biosynthesis and secretion of lysosomal cathepsin L in rat hepatocytes seem to be analogous to those of the major excreted protein of transformed mouse fibroblasts [S. Gal, M. C. Willingham, and M. M. Gottesman (1985) J. Cell Biol. 100, 535-544] and the mouse cysteine proteinase of activated macrophages [D.A. Portnoy, A. H. Erickson, J. Kochan, J. V. Ravetch, and J. C. Unkeless (1986) J. Biol. Chem. 261, 14697-14703].     

Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.     

Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.     

In vitro biosynthesis of two human galactosyltransferase polypeptides

HeLa cell galactosyltransferase is synthesized as two precursor polypeptides of Mr = 45,000 and Mr = 47,000. The enzyme is present in the Golgi complex as a (mature) Mr = 54,000 glycoprotein. If cells are treated with tunicamycin, two precursor polypeptides are synthesized without N-linked oligosaccharides with molecular weights of 42,000 and 44,000, respectively. To investigate whether the two precursor polypeptides are synthesized on different mRNAs total RNA from HeLa cells was translated in a wheat germ cell-free system. Galactosyltransferase polypeptides were isolated by immunoprecipitation and compared to the polypeptides synthesized in vivo in the presence of tunicamycin. The two in vitro translated polypeptides co-migrate exactly with the polypeptides made in the cells in the presence of tunicamycin, indicating two different mRNAs for galactosyltransferase. The results also indicate that translocation of galactosyltransferase through the membrane of the rough endoplasmic reticulum is not followed by signal peptide cleavage.     

Synthesis, processing, and secretion of the Marek''s disease herpesvirus A antigen glycoprotein.

The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV-A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption.     

Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice

The Mr 63,000 membrane polypeptide (gp63) is one of the Leishmania receptors for host macrophages and has been shown to protect mice from infection. The gene encoding gp63, the major Mr 63,000 surface glycoprotein of L. major promastigotes, has been expressed as a fusion protein with the enzyme glutathione S- transferase encoded by the parasitic helminth Schistosoma japonicum. This fusion protein was recognized by polyclonal antibodies to the native Leishmania gp63 polypeptide. The insoluble gp63 fusion protein was purified by SDS-PAGE and electroelution and was used to raise antibodies in rabbits. These rabbit anti-gp63 antibodies recognized the fusion protein and the denatured parasite gp63 on immunoblots and by immunofluorescence on fixed promastigotes, but did not recognize the native molecule on live organisms. However, antibodies raised against native promastigote glycoproteins, affinity purified on solid-phase gp63 fusion protein, recognized both native and denatured gp63, suggesting the presence of native determinants in the recombinant protein. The gp63 fusion protein did not protect mice of either healer or nonhealer phenotype from challenge infection with live promatigotes. The implications of these results for the engineering of recombinant DNA-produced molecular vaccines are discussed.     

A new negatively regulated acute-phase phosphoprotein synthesized by rat hepatocytes.

The effect of acute inflammation on the production of the major phosphorylated protein (PP63) excreted by rat hepatocytes was investigated. Both intra- and extracellular forms of the protein labelled with [32P]Pi, [3H]fucose and [35S]methionine were immunoprecipitated with monospecific polyclonal antibodies, and relative rates of PP63 synthesis were measured. The hepatocytes of acutely inflamed rats produced and excreted 85% less 32P- and 3H-labelled PP63 than did control cells. This decreased amount of PP63 did not result from an impairment in the phosphorylation or glycosylation processes or from a blockade in excretion, but rather was found to be due to extensive shut-off in biosynthesis of the protein as measured by [35S]methionine incorporation. Thus PP63 would appear to represent a new negatively regulated acute-phase protein.     

Biosynthesis of mosquito vitellogenin

Vitellogenin (Vg), the hemolymph precursor to the major yolk protein in mosquitoes, is synthesized in the fat body of blood-fed females. Mosquito Vg consists of two subunits with Mr = 200,000 and 66,000. Here, we demonstrate that both the Vg subunits are first synthesized as a single precursor. The identity of this Vg precursor was confirmed by immunoprecipitation with subunit-specific monoclonal antibodies. In cell-free translation of fat body poly (A)+ RNA, the Vg precursor had Mr = 224,000 which increased to 240,000 in the presence of canine pancreatic microsomal membranes. A precursor with Mr = 250,000 was immunoprecipitated in microsomal fractions isolated from rat bodies. With in vitro pulse labeling, the 250-kDa precursor could be detected in homogenates of fat bodies from blood-fed mosquitoes only during the first few hours accumulation of the Vg precursor was achieved by an in vitro stimulation of Vg synthesis in previtellogenic fat bodies cultured with an insect hormone, 20-hydroxyecdysone. The 250-kDa precursor was glycosylated and to a much lesser degree phosphorylated. Treatment of fat bodies with tunicamycin yielded the precursor with Mr = 226,000 which was neither glycosylated nor phosphorylated. The reduction in molecular mass of the 250-kDa Vg precursor and of both mature Vg subunits combined was similar after digestion with endoglycosidase H, indicating that glycosylation is completed prior to cleavage of the Vg precursor. In vitro pulse-chase experiments revealed rapid proteolytic cleavage of the 250-kDa precursor to two polypeptides with Mr = 190,000 and 62,000 which transformed into mature Vg subunits of 200- and 66-kDa as the last step prior to Vg secretion. This last step in Vg processing was inhibited by an ionophore, monensin, and therefore occurred in the Golgi complex. Sulfation as an additional, previously unknown, modification of mosquito Vg was revealed by the incorporation of sodium [35S]sulfate into both Vg subunits. Since sulfation of Vg was predominantly blocked by monensin, the final maturation of Vg subunits in the Golgi complex is, at least in part, due to this modification.     

Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture.

Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells.     

Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.     

Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.     

Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts.

The precursor protein to the chick corneal keratan sulfate proteoglycan was identified by immunoprecipitation with antiserum to its core protein from lysates of [35S]methionine-pulsed corneas and corneal fibroblasts in cell culture. Antiserum to the keratan sulfate proteoglycan immunoprecipitated a doublet of Mr 52,000 and 50,000 and minor amounts of a Mr 40,000 protein from pulsed corneas. Pulse-chase experiments, which permitted the conversion of the precursor proteins to proteoglycans and digestion of the glycosaminoglycans on immunoprecipitated proteoglycans with keratanase or chondroitinase ABC, showed that the Mr 52,000-50,000 doublet was converted to a keratan sulfate proteoglycan and the Mr 40,000 protein was converted to a chondroitin sulfate proteoglycan. Chick corneal fibroblasts in cell culture primarily produced the smaller (Mr50,000) precursor protein, and in the presence of tunicamycin the precursor protein size was reduced to Mr35,000, which indicates that the core protein contains approximately five N-linked oligosaccharides. Pulse-chase experiments with corneal fibroblasts in culture showed that the precursor protein was processed and secreted into the medium. However, its sensitivity to endo-beta-galactosidase and resistance to keratanase indicate that the precursor protein was converted to a glycoprotein with large oligosaccharides and not to a proteoglycan. This suggests that, although the precursor protein for the proteoglycan is produced in cultured corneal fibroblasts, the sulfation enzymes for keratan sulfate may be absent.     

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.     

The effects of glucagon, phenylephrine and insulin on the phosphorylation of cytoplasmic, mitochondrial and membrane-bound proteins of intact liver cells from starved rats.

1. The effects of glucagon, insulin and phenylephrine on the phosphorylation of cytoplasmic, mitochondrial and membrane proteins were studied in intact hepatocytes from 24 h-starved rats incubated with [32P]Pi. A rapid cell-fractionation technique was used, followed by radioautography of the proteins separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. Glucagon consistently caused a significant increase in the phosphorylation of four readily separable cytoplasmic phosphoproteins, of Mr 93000, 50000, 46000 and 20000, and a decrease in phosphorylation of a phosphoprotein of Mr 22000. Phosphorylation of the protein of Mr 46000 was also enhanced by both phenylephrine and insulin, and that of Mr 93000 by phenylephrine. 3. The phosphoprotein of Mr 22000 was not precipitated by boiling for 5 min, and had a mobility identical with that of similar protein whose phosphorylation is enhanced in the adipocyte by insulin [Belsham & Denton (1980) Biochem. Soc. Trans. 8, 382-383]. 4. Glucagon, but not phenylephrine or insulin, enhanced the phosphorylation of a mitochondrial protein of Mr 35000 and of four plasma- or microsomal-membrane proteins of Mr 50000, 30000, 23000 and 19000. 5. Mitochondria from glucagon-treated animals or hepatocytes phosphorylated a protein of Mr 30000 when incubated in vitro with [32P]Pi and ADP. Phosphorylation of this protein did not occur with mitochondria from control, phenylephrine- or insulin-treated cells. 6. The significance of these hormonally induced changes in protein phosphorylation is discussed.     

Influence of glycosylation on allelic and cell-specific Mr variation, receptor processing, and ligand binding of the human complement C3b/C4b receptor

The human complement C3b/C4b receptor (CR1), a single chain membrane glycoprotein of Mr approximately 200,000, has several cell-specific Mr variations as well as an allelic variation involving four phenotypes whose Mr values span a range of 90,000. We investigated the role of glycosylation in these structural variations and in receptor metabolism. In the human HL-60 promyelocytic leukemic cell line differentiated toward granulocytes or monocytes and in Epstein-Barr virus-transformed B lymphoblastoid cell lines of the common phenotype, CR1 is synthesized as a precursor of Mr = 222,000 that is converted into mature CR1s with differing Mr values. Endoglycosidase F treatment of mature CR1 from all these cell types produced the same lower Mr band. Additionally, the previously noted 5,000 higher Mr of CR1 from human peripheral granulocytes versus erythrocytes was abrogated by endoglycosidase F. Hence these cell-specific variations are due to differences in N-glycosylation. Lectin affinity chromatography shows that these N-linked oligosaccharides are mostly tri- and tetraantennary complex-type species with specific differences in fractionation patterns that correlate with their differing Mrs. In lymphoblastoid cell lines, the four allelic variants each have a precursor 6,000 lower in Mr than the respective mature CR1. In the presence of tunicamycin, each of the CR1 allelic products is 25,000 lower in Mr than the glycosylated receptor. The failure to radiolabel CR1 with [3H]glucosamine in the presence of tunicamycin indicates the lack of O-linked oligosaccharide on CR1. These data, taken together, strongly suggest that the CR1 polymorphism resides at the polypeptide level. Nonglycosylated CR1 synthesized in the presence of tunicamycin had twice the turnover rate of glycosylated CR1. The efficiency of surface membrane insertion and of ligand binding to hemolytically inactive C3 were markedly reduced for nonglycosylated CR1, suggesting that glycosylation is important for the proper expression of CR1 function.