Use of recombinant DNA derived human relaxin to probe the structure of the native protein

This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was equipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the complete amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (CysA10-CysA15, CysA11-CysB11, and CysA24-CysB23) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.     

cDNA sequence and deduced amino acid sequence of the precursor of the 37-kDa inner envelope membrane polypeptide from spinach chloroplasts. Its transit peptide contains an amphiphilic alpha-helix as the only detectable structural element

We present the nucleotide sequence and the deduced amino acid sequence of a cDNA clone that encodes the entire precursor of the 37-kDa inner envelope membrane protein from spinach chloroplasts. The precursor protein consists of 344 amino acids (Mr 38,976). In vitro processing followed by radiosequence analysis of the in vitro transcribed and translated precursor protein revealed that its transit peptide consists of only 21 amino acid residues. The transit peptide has the potential to form an amphiphilic alpha-helix with a strong hydrophobic moment. It is speculated that this structural element represents an ancestral envelope-targeting domain. The in vitro synthesized precursor protein is directed to the chloroplasts and it is inserted into the envelope membrane in an ATP-dependent manner. The mature protein (323 amino acid residues, Mr 36,830) has a moderate hydrophobicity and contains only one membrane-spanning segment which is located at the C-terminus and possibly anchors the protein within the envelope membrane.     

Cell-free biosynthesis of multiple preprosomatostatins: characterization by hybrid selection and amino-terminal sequencing

In vitro translation of mRNA isolated from islets of Langerhans results in the synthesis of three major preprosomatostatins of Mr 19 000, 18 000, and 16 000, each of which can be resolved into several isoelectric forms [Warren, T. G., & Shields, D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3729-3733]. Here we present further characterization of the somatostatin precursors by (i) hybrid selection translation of specific preprosomatostatin mRNAs, (ii) in vitro proteolytic processing of the nascent preprosomatostatins synthesized from hybrid-selected mRNAs, (iii) comparison of their tryptic peptides, and (iv) partial amino-terminal sequence analysis of the signal peptide regions. Hybrid selection experiments using specific cDNA clones demonstrated which preprosomatostatin species corresponded to previously characterized precursor cDNAs [Hobart, P., Crawford, R., Shen, L. P., Picket, R., & Rutter, W. J. (1980) Nature (London) 288, 137-141]; thus, the polypeptide encoded by plasmid pLaS1 corresponds to one form of the Mr 18 000 preprosomatostatins while one form of the Mr 16 000 preprosomatostatins is encoded by pLaS2. Analysis of the tryptic peptides demonstrated that the Mr 16 000 molecule possessed the mature hormone sequence at the carboxyl terminus, as had been shown for the Mr 19 000 and 18 000 precursors. Partial NH2-terminal sequence analysis (a) confirmed the data from hybrid selection and (b) demonstrated that the Mr 18 000 precursor contained a signal peptide manifesting amino acid heterogeneity at certain positions in the signal peptides of each preprosomatostatin. It is suggested that this heterogeneity might account, in part, for variants of the preprosomatostatin molecules.     

Structure of a precursor to human pancreatic polypeptide

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.     

Secretion of a major phosphorylated glycoprotein by hepatocytes. Characterization of specific antibodies and investigations of the processing, excretion kinetics, and phosphorylation

Isolated rat hepatocytes secreted a major phosphorylated glycoprotein (PP63) with apparent Mr = 63,000 and isoelectric point ranging from 4.8 to 5.3. Specific antibodies were raised in a rabbit using material obtained from plasma as an antigen. The biosynthesis of PP63 was studied in vitro in a cell-free system and in intact hepatocytes incubated with or without tunicamycin. The mRNA translation product had a Mr = 43,000 and was of the same size as the major unglycosylated precursor found in intact cells. This precursor was rapidly processed into two major intracellular forms of Mr = 53,000 and 56,000. These species were insensitive to neuraminidase but susceptible to endoglycosidase H, indicating that they contained oligosaccharide side chains of the high mannose-type. Terminal glycosylation gave rise to the mature Mr = 63,000 protein that contained sialic acid and fucose. This species represented the exportable form of the protein and was the only one to be phosphorylated. The charge heterogeneity observed for the mature protein already existed in all the precursors, indicating that it could not be ascribed to sialylation or to phosphorylation. However, these covalent modifications were mainly responsible for the acidic character of PP63. PP63 secretion was altered by tunicamycin. Pulse-chase experiments showed that the phosphorylated glycoprotein was secreted according to kinetics similar to that described for other liver glycoprotein, with slower kinetics than albumin. Permanent phosphorylation did not appear mandatory for excretion since the dephosphorylated PP63 was excreted with an efficacy comparable to that of the phosphorylated protein. Phosphorylation of PP63 was shown to occur on a single tryptic peptide, at a serine residue.     

Presclerotized eggshell protein from the liver fluke Fasciola hepatica

Trematode parasites protect their eggs with a tough tanned eggshell. Eggshell precursor proteins are synthesized and stockpiled within the extensive vitellaria of the animal. A major eggshell precursor protein with an apparent molecular weight of 31,000 and pI of 7.4 was isolated from the vitellaria of Fasciola hepatica. This protein, which represents 6-7% of the total protein in mature Fasciola, is unique in containing rather high levels of the amino acid 3,4-dihydroxyphenylalanine (DOPA), i.e., 110 residues per 1000. Other prominent amino acids are glycine, aspartic acid, and lysine. A prominent DOPA-containing tryptic peptide derived from eggshell precursor protein has the sequence Gly-Gly-Gly-DOPA-Gly-Gly-DOPA-Gly-Lys. DOPA residues disappear during the maturation of the eggshell and by treatment in vitro with mushroom polyphenol oxidase. This disappearance may be related to the formation of cross-links in the eggshell protein.     

Cell-free synthesis of tryptophanase from Escherichia coli. Use of ribonucleic acid isolated from induced cells and a comparison of the product from a system employing ribosomes with that from one employing ribosomes and exogenous ribonucleic acid

1. Polyribosomes and RNA were isolated from cultures in which tryptophanase (EC 4.2.1.-) was induced. The polyribosomes were incubated under conditions of protein synthesis, in the presence of a radioactive amino acid and a post-ribosomal supernatant fraction obtained from repressed cells. The RNA preparations were incubated under conditions of protein synthesis in the presence of a radioactive amino acid and a supernatant fraction containing ribosomes from repressed cells. 2. The system was characterized and the synthesis of a radioactive protein with the same chromatographic properties as tryptophanase was demonstrated. This synthesis was shown to be time-dependent and required the presence of RNA from induced cultures, ribosomes and an energy supply; it was inhibited by chloramphenicol. 3. The maximum activity for the synthesis of this protein was found to be associated with 23S rRNA isolated from sucrose gradients. 4. The N-terminal amino acid of tryptophanase was labelled in the protein synthesized in this system but not in the protein synthesized by polyribosomes (without added RNA). Conversely, the C-terminal amino acid of tryptophanase was labelled in the polyribosome system but not in the RNA-containing system. 5. Tryptic digests of protein labelled in vitro were compared with those of tryptophanase. No labelled tryptic peptides were identified other than tryptophanase tryptic peptides. An analysis of the results implied that in the polyribosome system almost the complete tryptophanase subunit chain was labelled but that in the RNA-containing system these chains were incompletely synthesized. 6. Sucrose-gradient analysis of protein synthesized in the RNA-containing system suggested that it cannot be converted into structures with the same sedimentation properties as native tryptophanase. 7. The significance of these results for the assay of tryptophanase mRNA and for an understanding of the control of the translation of this mRNA in vivo is discussed.     

Initial events involved in the synthesis of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum

In Dictyostelium discoideum the lysosomal enzyme alpha-mannosidase is initially synthesized in vivo as a 140,000 Mr protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 Mr. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the alpha-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K alpha-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.     

Stimulation of rat uterine collagen synthesis by relaxin

Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.     

An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen

A Mr 23,000 Ag of the human trematode parasite, Schistosoma mansoni, has been identified by immunoscreening an adult worm cDNA library with antibody affinity purified on the Mr 23,000 to 25,000 integral membrane protein fraction of the parasite. This Ag is immunogenic in infected humans as well as in rabbits exposed to S. mansoni. The protein sequence of the Ag as deduced from cloned DNA sequences is 218 amino acids long and contains four putative transmembrane regions. Of particular significance, the Ag is strikingly similar, with respect to both amino acid sequence (36% identity) and putative domain structure to ME491, a human stage-specific melanoma-associated Ag.     

Total synthesis of human relaxin and human relaxin derivatives by solid-phase peptide synthesis and site-directed chain combination

Human relaxin, a two-chain protein hormone, was synthesized by solid-phase peptide synthesis in combination with a novel thiol-protecting group strategy whereby the three disulfide bonds could be synthesized sequentially and without error. The final product was shown to be homogeneous by reversed-phase high performance liquid chromatography and electrophoresis and had the correct amino acid composition and sequence. Tryptic digestion and peptide mapping of the synthetic relaxin by reversed-phase high performance liquid chromatography resulted in a pattern identical with that produced by standard tryptic relaxin fragments synthetized by different methods. Three human relaxin derivatives containing oxidized methionine, formyltryptophan, and bis[B13,B17-citrulline]-relaxin, were produced and their biological activity and structural similarity to human relaxin was assessed. All derivatives, except those containing modified tryptophan residues, showed indistinguishable circular dichroic spectra, indicating that the modifications did not cause significant structural changes. However, only human relaxin and the tryptophan- and methionine-protected relaxin derivatives showed bioactivity. The derivative in which the two arginines in positions B13 and B17 had been replaced by the uncharged isosteric amino acid citrulline were biologically inactive. This observation confirms preliminary studies (Büllesbach, E. E. and Schwabe, C. (1988) Int. J. Pept. Protein Res. 32, 361-367) that suggested that these two conserved arginines located in the midregion of the relaxin B chain are essential for the function of the hormone.     

Primary structure of rat brain prostaglandin D synthetase deduced from cDNA sequence

The amino acid sequence of rat brain prostaglandin D synthetase (Urade, Y., Fujimoto, N., and Hayaishi, O. (1985) J. Biol. Chem. 260, 12410-12415) was determined by a combination of cDNA and protein sequencing. cDNA clones specific for this enzyme were isolated from a lambda gt11 rat brain cDNA expression library. Nucleotide sequence analyses of cloned cDNA inserts revealed that this enzyme consisted of a 564- or 549-base pair open reading frame coding for a 188- or 183-amino acid polypeptide with a Mr of 21,232 or 20,749 starting at the first or second ATG. About 60% of the deduced amino acid sequence was confirmed by partial amino acid sequencing of tryptic peptides of the purified enzyme. The recognition sequence for N-glycosylation was seen at two positions of amino acid residues 51-53 (-Asn-Ser-Ser-) and 78-80 (-Asn-Leu-Thr-) counted from the first Met. Both sites were considered to be glycosylated with carbohydrate chains of Mr 3,000, since two smaller proteins with Mr 23,000 and 20,000 were found during deglycosylation of the purified enzyme (Mr 26,000) with N-glycanase. The prostaglandin D synthetase activity was detected in fusion proteins obtained from lysogens with recombinants coding from 34 and 19 nucleotides upstream and 47 and 77 downstream from the first ATG, indicating that the glycosyl chain and about 20 amino acid residues of N terminus were not essential for the enzyme activity. The amino acid composition of the purified enzyme indicated that about 20 residues of hydrophobic amino acids of the N terminus are post-translationally deleted, probably as a signal peptide. These results, together with the immunocytochemical localization of this enzyme to rough-surfaced endoplasmic reticulum and other nuclear membrane of oligodendrocytes (Urade, Y., Fujimoto, N., Kaneko, T., Konishi, A., Mizuno, N., and Hayaishi, O. (1987) J. Biol. Chem. 262, 15132-15136) suggest that this enzyme is a membrane-associated protein.     

B-chain sequence and in situ hybridization of the rabbit placental relaxin-like gene product.

We reported that the nucleotide sequence of a cDNA generated from rabbit placental poly(A)(+) RNA using porcine preprorelaxin primers was identical to SQ10, a product of squamous differentiated tracheal epithelial cells. However, these results did not confirm that SQ10 was the biologically active rabbit relaxin that had been isolated previously yet not sequenced. In this study, a 7-kDa protein isolated from rabbit placentas exhibited relaxin bioactivity and cross-reacted with a porcine relaxin antiserum. A partial amino acid sequence of this protein revealed a sequence identical to that of SQ10. Although the amino acid sequence of the putative relaxin receptor-binding domain found in the B chain of relaxin was modified in SQ10 from CGRDYVR to CRNDFVR, the placental protein was bioactive. These results suggest that SQ10 is the rabbit relaxin. In situ hybridization, using an SQ10 riboprobe, indicated radiolabeling in the syncytiotrophoblast cells of the rabbit placenta. The pattern of labeling corresponded with the immunohistochemical staining for relaxin observed with use of a porcine relaxin antiserum. These results indicate that the syncytiotrophoblast cells are a site of synthesis for SQ10 and that the immunostaining is not solely the result of sequestering SQ10 through receptor-mediated endocytosis. A potential role for relaxin in implantation is discussed.     

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcyctis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.     

Regulation of protein synthesis in eukaryotes. Eukaryotic initiation factor eIF-2 and eukaryotic recycling factor eRF from neuroblastoma cells

eIF-2 purified from neuroblastoma cells consists of three subunits, which appear to be of molecular weight identical to those of the subunits of rabbit reticulocyte eIF-2. A protein fraction has been isolated from neuroblastoma cells with characteristics similar to eRF from reticulocytes: stimulation of amino acid incorporation in a hemin-deprived reticulocyte lysate, the removal of GDP from eIF-2-GDP complexes, a 4-5-fold stimulatory effect in a two-step reaction measuring 40 S preinitiation complex formation and a 3-3.5-fold stimulation in the methionyl-puromycin synthesis. In the methionyl-puromycin-synthesizing system phosphorylated eIF-2 is not responsive to the addition of this fraction from neuroblastoma cells. The protein fraction contains eRF which seems to be similar to the eRF isolated from Ehrlich ascites tumor cells and somewhat distinct from the reticulocyte factor. Incubation of neuroblastoma cell lysate in the presence of [gamma-32P]ATP results in the phosphorylation of a protein of Mr 36 000, migrating on SDS-polyacrylamide gels to the position of eIF-2 alpha. This protein is also phosphorylated in vitro by HRI from reticulocytes. These results may reflect a common underlying principle for the quantitative regulation of protein synthesis in eukaryotic cells.     

Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2

The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.     

Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries

To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.     

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 of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells

Tyr(P)-containing proteins were purified from extracts of insulin-treated rat hepatoma cells (H4-II-E-C3) by antiphosphotyrosine immunoaffinity chromatography. Two major insulin-stimulated, Tyr(P) proteins were recovered: an Mr 95,000 protein (identified as the insulin receptor beta subunit by its immunoprecipitation by a patient-derived anti-insulin receptor serum and several anti-insulin receptor (peptide) antisera) and an Mr 180,000 protein (which was unreactive with all anti-insulin receptor antibodies). After purification and tryptic digestion of the Mr 95,000 protein, tryptic peptides containing Tyr(P) were purified by sequential antiphosphotyrosine immunoaffinity, reversed-phase, anion-exchange chromatography. The partial amino acid sequence obtained by gas- and solid-phase Edman degradation was compared to the amino acid sequence of the intracellular extension of the rat insulin receptor deduced from the genomic sequence. Approximately 80% of all beta subunit [32P]Tyr(P) resides on two tryptic peptides: 50-60% of [32P]Tyr(P) is found on the tryptic peptide Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Arg from the tyrosine kinase domain, which is recovered mainly as the double phosphorylated species (predominantly in the form with Tyr(P) at residues 3 and 7 from the amino terminus; the remainder with Tyr(P) at residues 3 and 8), with 10-15% as the triple phosphorylated species. A second tryptic peptide is located near the carboxyl terminus, contains 2 tyrosines, and has the sequence, Thr-Tyr-Asp-Glu-His-Ile-Pro-Tyr-Thr-; this contains 20-30% of beta subunit [32P]Tyr(P) and is identified primarily in a double phosphorylated form. Approximately 10% of beta subunit [32P]Tyr(P) resides on an unidentified tryptic peptide of Mr 4,000-5,000. The insulin-stimulated tyrosine phosphorylation of the insulin receptor in intact rat hepatoma cells thus involves at least 6 of the 13 tyrosine residues located on the beta subunit intracellular extension. These tyrosines are clustered in several domains in a distribution virtually identical to that previously found for partially purified human insulin receptor autophosphorylated in vitro in the presence of insulin. This multisite regulatory tyrosine phosphorylation is the initial intracellular event in insulin action.