Glycosylation of lipoprotein lipase in human subcutaneous and omental adipose tissues.

Human adipose tissues from the abdomen (subcutaneous), thigh (subcutaneous) and omentum were incubated for 2 h with [35S]methionine. Then glycosylation of lipoprotein lipase (LPL) was analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of the 35S-labeled lipase. Adipose tissues from the abdomen, thigh, and omentum all synthesized LPL subunits with Mr = 57,000 composed of two types of subunits. One type was partially endo H-sensitive yielding a product with Mr = 55,000, indicating that it had one endo H-resistant and one endo H-sensitive oligosaccharide chain. The other type of subunit was totally endo H-sensitive yielding a product with Mr = 52,000. Subcutaneous adipose tissues contained nearly equal amounts of partially and totally endo H-sensitive subunits of LPL, whereas omental adipose tissues contained mainly partially endo H-sensitive subunits of LPL.     

Biogenesis of the polymeric IgA receptor in rat hepatocytes. I. Kinetic studies of its intracellular forms

The polymeric IgA receptor (or secretory component [SC]) is a major biliary secretory protein in the rat. It was identified as an 80,000-mol-wt (80 K) glycoprotein by coprecipitation (with IgA) by anti-IgA antibodies (Sztul, E. S., K. E. Howell, and G. E. Palade, 1983, J. Cell Biol., 97:1582-1591) and was used as antigen to raise anti-SC antibodies in rabbits. Pulse labeling with [35S]cysteine in vivo, followed by the immunoprecipitation of solubilized total microsomal fractions with anti-SC sera, made possible the identification of three intracellular forms of SC (all apparently membrane proteins) and the definition of their kinetic and structural interrelations. At 5 min postinjection of [35S]cysteine, a major band of Mr 105,000 was maximally labeled. This peptide lost radioactivity concomitantly with the appearance of a radioactive doublet of Mr 116,000 and 120,000 at 15-30 min postinjection. Loss of radioactivity from 116K paralleled increased labeling of the 120K peptide which appears to be the mature form of the receptor. The 105K form was sensitive to endoglycosidase H which converted it to a 96K peptide. The 116K and 120K forms were resistant to endoglycosidase H but sensitive to endoglycosidase F which converts them to 96K and 100K forms, respectively. Taken together, these findings support the following conclusions: (a) All rat hepatic SC forms are the products of a single gene; (b) all SC forms are N-glycosylated; (c) the 116K form is the result of the terminal glycosylation of the 105K form; and (d) the 120K peptide is probably produced by modifications at other sites than its complex oligosaccharide chains.     

Evidence for stable homodimers and heterodimers of gamma-glutamyltranspeptidase subunits under protein-denaturing conditions

gamma-Glutamyltranspeptidase is synthesized as a core glycosylated propeptide (Mr 75,000) which is subsequently cleaved to yield a stable heterodimeric structure (subunit Mr 50,000 and 30,000). The propeptide represents an insignificant mass of the transpeptidase but higher molecular weight bands designated H1 (Mr 85,000) and H2 (Mr 100,000) are readily observed by protein staining or immunoblot analysis of the enzyme or crude membranes after SDS-polyacrylamide gel electrophoresis. Although H1 and H2 represent the predominant antigenic forms of transpeptidase in tissues which exhibit relatively low specific enzyme activity, neither their structure nor their physiological function is known. In order to determine the relationship between H1 and H2, and the large (L) and small (S) subunits of the transpeptidase, individual bands (H1, H2, L and S) of the purified renal enzyme were cut from a Coomassie-stained SDS gel, eluted and re-electrophoresed. Isolated S produced S and dimers of S (Mr 60,000), while isolated L produced L and dimers of L corresponding to H2. Equivalent mixtures of L and S also produced H1. Utilizing IgG affinity-purified against either L or S, immunoblot analysis confirmed that H2 is a dimer of L, and H1 is a heterodimer of L and S. However, monoclonal IgG which recognizes both transpeptidase propeptide and native heterodimer did not react with H1. Thus, it is clear that isolated L and S can form and maintain unique dimeric structures during SDS-polyacrylamide gel electrophoresis. With this information it should now be possible to ascertain the basis for the apparent predominance of H1 and H2 in non-renal tissues.     

Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine.

The effect of castanospermine (CSTP), an inhibitor of glucosidase I, on processing, activity, and secretion of lipoprotein lipase was studied in 3T3-L1 adipocytes. Processing was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase from cells incubated 1-2 h with [35S]methionine. Lipoprotein lipase in untreated cells consisted of two groups of subunits, M(r) = 55,000-58,000 and M(r) = 53,000-55,000. The heavier subunits were endo H-resistant, whereas the others were either totally or partially endo H-sensitive. The lipase secreted by untreated cells contained primarily endo H-resistant subunits. Immunofluorescent studies showed that lipoprotein lipase accumulated in Golgi in untreated cells. CSTP, 100 micrograms/ml for 18 h, decreased intracellular lipase activity by 80% and decreased secretion of lipase activity by 91%. Most of the lipase subunits in CSTP-treated cells were totally endo H-sensitive with M(r) = 57,000, some were partially endo H-sensitive, and a trace was endo-H resistant. Totally endo H-sensitive subunits in CSTP-treated cells had a M(r) 2,000-4,000 larger than that in untreated cells, indicating impaired trimming of sugar residues from oligosaccharide chains of the lipase in CSTP-treated cells. The small amount of lipase secreted by CSTP-treated cells consisted primarily of partially endo H-sensitive subunits, with one sensitive and one resistant chain per subunit. Immunofluorescent studies showed that lipoprotein lipase was excluded from Golgi in CSTP-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle

We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.     

The identification of two subcellular sites for cleavage of gamma-glutamyltranspeptidase propeptide

In order to determine the subcellular site(s) of rat renal gamma-glutamyltranspeptidase propeptide cleavage labeled immunoprecipitates were obtained from preparations of either intracellular membranes or brush border membrane vesicles. Heterodimer accounts for 25% of the label associated with transpeptidase in intracellular membranes from 5 to 40 min postinjection of [35S]methionine, consistent with a cotranslational cleavage of propeptide in the endoplasmic reticulum. Labeled propeptide and heterodimer appear in the brush border membrane fraction between 20-30 min postinjection and accumulate for 1 h and 4h, respectively. Subsequently, the propeptide disappears with a half-life of 1 h while the heterodimer is relatively stable. These results confirm our previous proposal for two distinct subcellular sites for transpeptidase propeptide cleavage (Capraro, M.A. and Hughey, R.P. (1983) FEBS Lett. 157, 139-143).     

Purification and characterization of bovine lactoferrin from secretions of the involuting mammary gland: identification of multiple molecular weight forms

1. Lactoferrin was isolated from bovine mammary secretions collected during the nonlactating period. 2. A method utilizing heparin-agarose affinity chromatography was more efficient for purifying lactoferrin than a method including gel filtration, ion exchange chromatography and a second gel filtration. 3. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the purified lactoferrin was composed of two protein bands of apparent mol. wt. of 83,000 and 87,000. 4. Digestion with endoglycosidase H resolved the lactoferrin into two lower mol. wt. bands of 78,000 and 81,000. 5. The biochemical differences between the forms of lactoferrin are not exclusively due to differences in endoglycosidase H-sensitive oligosaccharide composition.     

Retention of phytohemagglutinin with carboxyterminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum

Soluble proteins that reside in the lumen of the endoplasmic reticulum are known to have at their carboxyterminus the tetrapeptides KDEL or HDEL. In yeast and mammalian cells, these tetrapeptides function as endoplasmic reticulum (ER)-retention signals. To determine the effect of an artificially-introduced KDEL sequence at the exact carboxyterminus of a plant secretory protein, we modified the gene of the vacuolar protein phytohemagglutinin-L (PHA) so that the amino-acid sequence would end in LNKDEL rather than LNKIL, and expressed the modified gene in transgenic tobacco with a seed-specific promoter. Analysis of the glycans of PHA showed that most of the control PHA had one endoglycosidase H-sensitive and one endoglycosidase H-resistant glycan, indicating that it had been processed in the Golgi complex. On the other hand, a substantial portion of the PHA-KDEL (about 75% at mid-maturation and 50% in mature seeds) had two endoglycosidase H-sensitive glycans. Phytohemagglutinin with two endoglycosidase H-sensitive glycans is normally found in the ER. Using immunocytochemistry we found that a substantial portion of the PHA-KDEL was present in the ER or accumulated in the nuclear envelope while the remainder was found in the protein storage vacuoles (protein bodies). We interpret these data to indicate that carboxyterminal KDEL functions as an ER retention-retardation signal and causes protein to accumulate in the nuclear envelope as well as in the ER. The incomplete ER retention of this protein which is modified at the exact carboxyterminus may indicate that structural features other than carboxyterminal KDEL are important if complete ER retention is to be achieved.Mention of trademark, proprietary product, or vendor, does not constitute a guarantee or warrenty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations endoH endoglycosidase H - ER endoplasmic reticulum - Mr relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TBST Tris-buffered saline containing Tween 20 We thank Debra Donaldson for her contribution to the PHA gene constructions. This work has been supported by grants from the National Science Foundation (Cell Biology) and the Department of Energy (DE-FG03-86ER13497) to Maarten J. Chrispeels. The assistance of the staff of the Electron Microscope Laboratory, USDA, Beltsville is gratefully acknowledged.     

Glycosylation and secretion of surfactant-associated glycoprotein A

Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.     

Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice

Combined lipase deficiency (cld) is a recessive mutation which causes a severe deficiency of lipoprotein lipase and hepatic lipase activities and lethal hypertriacylglycerolemia within 3 days in newborn mice. The effect of this genetic defect on lipoprotein lipase was studied in primary cultures of brown adipocytes derived from tissue of newborn mice. Cells cultured from cld/cld mice replicated, accumulated triacylglycerol, and differentiated into adipocytes at normal rates. Lipoprotein lipase activity in unaffected cells was detectable on Day 0 of confluence and increased to 1.3 units/mg DNA by Day 6, while that in cld/cld cells was less than 4% of that in unaffected cells on Days 4-6. Unaffected cells released 1.2% of their lipase activity in 30 min in the absence of heparin, and 11% in 10 min in the presence of heparin, whereas cld/cld cells released no lipase activity. cld/cld cells contained 2-3 times as much lipoprotein lipase protein as unaffected cells, and released no lipase protein to the medium. Immunofluorescent lipoprotein lipase was not detectable in unaffected adipocytes unless lipase secretion was blocked with monesin, causing retention of the lipase in Golgi. cld/cld adipocytes, in contrast, contained immunofluorescent lipoprotein lipase distributed in a diffuse reticular pattern, indicating retention of lipase in endoplasmic reticulum. Lipoprotein lipase immunoprecipitated from cells incubated 1-3 h with [35S]methionine was digested with or without endoglycosidase H (endo H) or F, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoprotein lipase in unaffected cells (Mr = 56,000-58,000) consisted of three glycosylated forms, of which the most prevalent was endo H-resistant, the next was totally endo H-sensitive, and the least was partially endo H-sensitive. In contrast, lipoprotein lipase in cld/cld cells (Mr = 56,000) consisted of a single, totally endo H-sensitive form. Lipoprotein lipase in both groups of cells contained two oligosaccharide chains. Chromatography studies with heparin-Sepharose indicated that at least some of the lipoprotein lipase in cld/cld cells was dimerized. The findings demonstrate that brown adipocytes cultured from cld/cld mice synthesize lipoprotein lipase with two high mannose oligosaccharide chains, but it is inactive and retained in endoplasmic reticulum. Whether the cld mutation affects primarily processing of oligosaccharide chains of lipoprotein lipase in endoplasmic reticulum, transport of the lipase from the reticulum, or some other process, is to be resolved.     

Complexes of rat alpha 1-macroglobulin and subtilisin are endocytosed by parenchymal liver cells.

Rat alpha 1-macroglobulin was isolated from plasma. Gel electrophoresis of the denatured and reduced protein showed two bands, with Mr values of 163 000 and 37 000. The large subunit contained an autolytic site. This subunit was also split after reaction of the macroglobulin with trypsin. Electron microscopy showed that the macroglobulin changed towards a more compact conformation after reaction with this proteinase. Subtilisin, or alpha 1-macroglobulin, was labelled with a sucrose-containing radio-iodinated group that stays in lysosomes after endocytosis and breakdown of the tagged protein. After intravenous injection into rats, alpha 1-macroglobulin was cleared from plasma with first-order kinetics, showing a half-life of about 9 h, whereas complexes of alpha 1-macroglobulin and subtilisin were cleared with half-lives of only 3 min. Liver contained about 60% of the label at 30 min after injection of complexes. About 90% of the liver radioactivity was found in parenchymal cells isolated after perfusion of the liver with a collagenase solution. Subcellular fractionation indicated a lysosomal localization of the complexes. We conclude that endocytosis by parenchymal liver cells is the major cause of the rapid clearance of alpha 1-macroglobulin-proteinase complexes from plasma.     

Characterization of a Platelet-Derived Growth Factor Receptor on Swiss 3T3 Cells by Affinity Crosslinking

Abstract

Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205′000 and Mr 190′000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable pazidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190′000 band represents a Mr 175′000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205′000 species the same Mr 175′000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150′000, while pretreatment with neuraminidase resulted in a shift of the Mr 205′000 and 190′000 bands by 5′000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr β 135′000 proteinaceous core and a Mr β 40′000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37°C preformed ¹²⁵I-PDGF receptor complexes disappear from the cell surface with a t_1/2 β 8 min.     

Differential processing and turnover of the oncogenically activated neu/erb B2 gene product and its normal cellular counterpart

In nontransformed DHFR/G-8 cells (NIH 3T3 cells transfected with normal rat neu gene), the normal neu gene product was initially synthesized as a 170-kDa protein bearing endoglycosidase H-sensitive oligosaccharide chains and was then processed to a 175-kDa mature form with endoglycosidase H-resistant, endoglycosidase F-sensitive oligosaccharide chains. Most of this 175-kDa mature form appeared on the cell surface 2 h following synthesis and showed a half-life of approximately 3 h. In the presence of a growth factor(s) partially purified from bovine kidney, the half-life of this 175-kDa normal neu gene product was shortened to less than 30 min. In B104-1-1 cells (NIH 3T3 cells transfected with neu gene activated oncogenically by a point mutation that changes a valine residue to a glutamic acid residue in the putative transmembrane region), the oncogenically activated neu gene product was also synthesized as a 170-kDa precursor with endoglycosidase H-sensitive oligosaccharide chains. However, this 170-kDa precursor diminished very fast and was only partially processed to a 185-kDa mature form which exhibited a half-life of less than 30 min. The 185-kDa activated neu gene product possessed an unidentified post-translational modification in addition to N-linked oligosaccharide chains. Both the precursor and mature forms of the mutationally activated neu gene product showed increased tyrosine-specific phosphorylation as compared with those of their normal counterparts in DHFR/G-8 cells. The mutationally activated neu gene product in B104-1-1 cells shared several features which have been reported previously for the ligand-activated platelet-derived growth factor receptor in v-sis- or c-sis-transformed cells. These properties include: 1) accelerated turnover of the precursor and mature forms compared with the rates of turnover of its normal counterparts, 2) insensitivity of this rapid turnover to lysosomotropic amines, and 3) increased in vivo tyrosine-specific phosphorylation of both the precursor and mature forms. These findings suggest that the mutationally activated neu gene product may transform the cells by mimicking ligand-induced activation.     

The sodium channel from rat brain. Separation and characterization of subunits

Procedures are described for separation of the alpha, beta 1, and beta 2 subunits of the voltage-sensitive sodium channel from rat brain by gel filtration in sodium dodecyl sulfate (SDS) before and after reduction of intersubunit disulfide bonds or by preparative SDS-gel electrophoresis. Partial proteolytic maps of the SDS-denatured subunits indicate that they are nonidentical polypeptides. They are all heavily glycosylated and contain complex carbohydrate chains that bind wheat germ agglutinin. The apparent molecular weights of the separated subunits were estimated by gradient SDS-gel electrophoresis, by Ferguson analysis of migration in SDS gels of fixed acrylamide concentration, or by gel filtration in SDS or guanidine hydrochloride. For the alpha subunit, SDS-gel electrophoresis under various conditions gives an average Mr of 260,000. Gel filtration methods give anomalously low values. Removal of carbohydrate by sequential treatment with neuraminidase and endoglycosidase F results in a sharp protein band with apparent Mr = 220,000, suggesting that 15% of the mass of the native alpha subunit is carbohydrate. Electrophoretic and gel filtration methods yield consistent molecular weight estimates for the beta subunits. The average values are: beta 1, Mr = 36,000, and beta 2, Mr = 33,000. Deglycosylation by treatment with endoglycosidase F, trifluoromethanesulfonic acid, or HF yields sharp protein bands with apparent Mr = 23,000 and 21,000 for the beta 1 and beta 2 subunits, respectively, suggesting that 36% of the mass of the native beta 1 and beta 2 subunits is carbohydrate.     

Asparagine-linked glycosylation of cytochrome b558 large subunit varies in different human phagocytic cells

Cytochrome b558, an essential component of the respiratory burst of phagocytic cells, is the terminal electron donor to molecular oxygen that results in the formation of superoxide anion (O2-.). It is an integral membrane heterodimer that in neutrophils consists of a 22-kDa small subunit and a highly glycosylated 91-kDa large subunit. Identical core proteins often differ in glycosylation in different cell types and with some membrane glycoproteins, the glycosylation state may markedly affect function. In the present study, antisera reactive with cytochrome b558 large subunit was used for immunoblot analysis of the glycosylation pattern of this subunit from different types of phagocytic cells. Striking variability in the apparent m.w. of this broadly banding subunit was detected in five different phagocytic cell types (neutrophils 78,000 to 93,000; eosinophils 74,000 to 115,000; monocytes 82,000 to 99,000; dibutyryl cyclic AMP-induced HL-60 cells 79,000 to 103,000; dimethyl sulfoxide-induced HL-60 cells 77,000 to 110,000). However, after complete cleavage of N-linked oligosaccharides with endoglycosidase F, the core peptide of cytochrome b558 large subunit from these different cell types had the same Mr (58,000). Inhibition of N-glycosylation with tunicamycin in differentiating HL-60 cells resulted in the synthesis of immunoreactive protein of the same m.w. and banding pattern as seen after endoglycosidase F cleavage. These tunicamycin treated cells retained some capacity to generate superoxide anion when stimulated with PMA. We conclude that the identity of the N-linked oligosaccharides of the cytochrome b558 large subunit differ in various phagocytic cells. All N-linked glycans on cytochrome b558 in all cell types examined were of the complex type as defined by resistance to endoglycosidase H cleavage. N-linked glycosylation of the cytochrome b558 large subunit may not be essential for activation of the respiratory burst.     

In vitro biosynthesis and membrane insertion of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase consists of two polypeptide chains anchored to the kidney brush-border membrane only through a short hydrophobic domain near the NH2-terminal end of the heavy subunit. The two subunits were reported to derive from a single polypeptide precursor by tissue labeling experiments. We have investigated the first steps of GGT biosynthesis and processing in a cell-free system. mRNA was prepared from kidney and enriched in specific sequences by a preparative gel electrophoresis. In vitro translation resulted in the synthesis of a single polypeptide (Mr = 63,000) specifically immunoprecipitated by antibodies raised against the mature dimeric enzyme. Incubation with microsomal membranes resulted in the appearance of a glycosylated form of the propeptide (Mr = 78,000). This latter form was cotranslationally segregated into microsomes and was sensitive to endoglycosidase H. Purified Escherichia coli leader peptidase did not process the primary gamma-glutamyl transpeptidase chain. This ectoprotein therefore appears to be inserted in the phospholipid bilayer without cleavage of a signal peptide, similar to most integral membrane proteins so far studied.     

Post-translational modification of the protein-synthesis initiation factor eIF-4D by spermidine in rat hepatoma cells.

The structural characteristics and glycoprotein nature of the human growth hormone (hGH) receptor in cultured lymphocytes (IM-9 cell line) were studied with the use of a bifunctional reagent (disuccinimidyl suberate) to couple 125I-hGH covalently to intact cells. After cross-linking, the hormone-receptor complexes were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A single band of Mr 140,000 was identified under reducing conditions. The labelling of this band was blocked by unlabelled hGH but not by insulin, ovine prolactin, bovine or ovine growth hormones. The Mr 140,000 band was immunoprecipitated by either anti-hGH antibody or by a monoclonal antibody against rat liver growth hormone receptor. In the absence of reductant two major bands of Mr 270,000 and 140,000 were found. On two-dimensional gel electrophoresis, with the first dimension in the absence of reductant and the second in its presence, the Mr 270,000 complex generated the Mr 140,000 band. The nature of the oligosaccharide chains of the receptor was studied by treatment with different glycosidases. The electrophoretic mobility of the Mr 140,000 receptor complex was markedly increased after digestion with endoglycosidase F but showed no or little change after digestion with endoglycosidase H. The Mr 140,000 band was also sensitive to neuraminidase treatment. In addition the 125I-hGH-receptor complex was adsorbed by immobilized wheat germ agglutinin and to a smaller extent by immobilized concanavalin A, lentil lectin, ricin I and ricin II. In conclusion, taking into account that hGH is a Mr 22,000 polypeptide, the binding subunit of the GH receptor in human IM-9 lymphocytes has an Mr of approx. 120,000. The native receptor may exist as a homodimer of the binding subunit formed by disulphide bonds. Furthermore, the GH receptor subunit contains asparagine N-linked type of oligosaccharide chains. Most, if not all, of these chains are of the complex type and appear to be sialylated whereas no high-mannose type chains are detectable in the mature form of the receptor.     

Further characterization of human eosinophil peroxidase.

The large and the small subunits (Mr 50 000 and 10 500 respectively) of human eosinophil peroxidase were isolated by gel filtration under reducing conditions. The subunits were very strongly associated but not apparently cross-linked by disulphide bridges. During storage, the large subunit tended to form aggregates, which required reduction to dissociate them. Amino acid analysis of the performic acid-treated large subunit showed the presence of 19 cysteic acid residues. The small subunit of eosinophil peroxidase had the same Mr value as the small subunit of myeloperoxidase. However, although these subunits have very similar amino acid compositions, they showed different patterns of peptide fragmentation after CNBr treatment. The carbohydrate of eosinophil peroxidase seemed associated exclusively with the large subunit and comprised mannose (4.5%, w/w) and N-acetylglucosamine (0.8%, w/w). The far-u.v.c.d. spectrum of the enzyme indicated the presence of relatively little ordered secondary structure.