Identification and processing of proglucagon in pancreatic islets.

Immunoprecipitation and tryptic peptide analysis of newly synthesized proteins from rat islets have identified an 18,000 molecular weight (MW) protein as proglucagon. Conversion of this precursor was kinetically similar to the conversion of proinsulin and resulted in the formation of both pancreatic glucagon and a 10,000-MW protein lacking this hormonal sequence.     

A signal sequence receptor in the endoplasmic reticulum membrane

Protein translocation across the endoplasmic reticulum (ER) membrane is triggered at several stages by information contained in the signal sequence. Initially, the signal sequence of a nascent secretory protein upon emergence from the ribosome is recognized by a polypeptide of relative molecular mass 54,000 (Mr54K) which is part of the signal recognition particle (SRP). Binding of SRP may induce a site-specific elongation arrest of translation in vitro. Attachment of the arrested translation complex to the ER membrane is mediated by the SRP-receptor (docking protein) and is accompanied by displacement of the SRP from both the ribosome and the signal sequence. We have investigated the fate of the signal sequence following the disengagement of SRP and its receptor by a crosslinking approach. We report here that the signal sequence of nascent preprolactin, after its release from the SRP, interacts with a newly discovered component, a signal sequence receptor (SSR), which is an integral, glycosylated protein of the rough ER membrane (Mr approximately 35K).     

The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle

Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides.     

Antisera to a synthetic peptide of the sis viral oncogene product recognize human platelet-derived growth factor

It has recently been reported that the sequences of the sis oncogene of simian sarcoma virus (SSV) and of human platelet-derived growth factor (PDGF) are very similar, establishing the most solid link yet between the mitogenic actions of growth factors and the transforming proteins of retroviruses. To investigate molecular mechanisms of transformation I have produced antisera against synthetic peptides corresponding to segments of the protein sequences predicted by the nucleotide sequences of viral oncogenes. Applying this approach to the case of sis and PDGF, I report here the results of probing outdated human platelets with an antiserum directed against a synthetic peptide representing residues 139-155 of the predicted sequence of the SSV transforming protein, p28sis (ref. 3). I detected peptides of apparent molecular weights (MWs) 30,000 to 31,000 (30-31K) and 16-18K, which correspond to the apparent molecular weights of nonreduced and reduced PDGF. In addition, a peptide of MW 21,000 was detected in platelets and a protein of MW 56,000 was detected in SSV-infected marmoset cells.     

Homology of 54K protein of signal-recognition particle, docking protein and two E. coli proteins with putative GTP-binding domains

Most proteins exported from mammalian cells contain a signal sequence which mediates targeting to and insertion into the membrane of the endoplasmic reticulum (ER). Involved in this process are the signal-recognition particle (SRP) and docking protein (DP), the receptor for SRP in the ER membrane. SRP interacts with the signal sequence on nascent polypeptide chains and retards their further elongation, which resumes only after interaction of the arrested ribosomal complex with the docking protein. SRP is a ribonucleoprotein particle comprising a 7S RNA and six polypeptides with relative molecular masses (Mr) of 9,000 (9K) 14K, 19K, 54K, 68K and 72K (ref. 1). The 9K and 14K proteins are essential for elongation arrest and the 68K-72K heterodimer is required for docking to the ER membrane. The 54K protein binds to the signal sequence when it emerges from the ribosome. Docking protein consists of two polypeptides, a 72K alpha-subunit (DP alpha) and a 30K beta-subunit (DP beta). No components structurally homologous to SRP and docking protein have yet been found in yeast or Escherichia coli. To understand the molecular nature of the interaction between the signal sequence and its receptor(s) we have characterized a complementary DNA coding for the 54K protein of SRP. Significant sequence homology was found to part of DP alpha and two E. coli proteins of unknown function. The homologous region includes a putative GTP-binding domain.     

Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide

Recent identification of natriuretic-diuretic activity in peptides isolated from human and rat atrial tissue implicates them in the control of extracellular fluid volume and electrolytic homeostasis. The presence of multiple forms of the peptides ranging from 3,000 to 13,000 molecular weight (MW) suggests they may all derive from the same precursor. The established amino acid sequence of alpha-human atrial natriuretic polypeptide (alpha- hANP ), a 28-residue peptide with potent natriuretic activity, provided the means to elucidate the structure of the precursor for alpha- hANP and the gene encoding it. Here we report the cloning and sequence analysis of the cDNA of human atrial mRNA encoding a precursor of alpha- hANP . The cDNA encodes gamma-human atrial natriuretic polypeptide (gamma- hANP ) of 13,000 MW, whose C-terminal 28 amino acid residues may be processed as alpha- hANP .     

Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta

Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.     

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27

Vasoactive intestinal polypeptide (VIP), a 28-amino acid peptide originally isolated from porcine duodenum, is present not only in gastrointestinal tissues but also in neural tissues, possibly as a neurotransmitter, and exhibits a wide range of biological actions (for example, relaxation of smooth muscle, stimulation of intestinal water and electrolyte secretion and release of insulin, glucagon and several anterior pituitary hormones). As the structure of porcine and bovine VIP shows several similarities to those of mammalian glucagon, secretin and gastric inhibitory peptide (GIP), VIP is considered to be a member of the glucagon-secretin family. Recently, we have found that VIP is synthesized from a precursor, pro-VIP (molecular weight (Mr) 17,500), in human neuroblastoma cells and that the primary translation product of the mRNA encoding VIP is prepro-VIP (Mr 20,000). In an attempt to elucidate the primary structure of the precursor, we have now cloned the DNA sequence complementary to the mRNA coding for human VIP and analysed the nucleotide sequence. The entire amino acid sequence of the precursor, deduced from the nucleotide sequence, indicates that the precursor protein contains not only VIP but also a novel peptide of 27 amino acids. The peptide, designated PHM-27, differs by only 2 amino acids from PHI-27, a peptide recently isolated from porcine intestine, and is also closely related in sequence to VIP.     

Most of the coding region of rat ACTH beta--LPH precursor gene lacks intervening sequences

The peptide hormones ACTH, beta-endorphin, alpha- and beta-melanotropin(MSH) and possibly gamma-MSH are synthesized in the pituitary gland by the processing of a 32,000-molecular weight (MW) polypeptide called proopiomelanocortin (POMC). The existence of a further precursor (pre form) to POMC containing an additional N-terminal 'leader' peptide has been suggested by analysis of the in vitro translation products of poly(A)-containing RNA from AtT-20 cells, a mouse ACTH-producing cell line of pituitary origin. Nakanishi et al. cloned and sequenced a cDNA copy of the bovine prePOMC mRNA. This sequence confirmed the known structure of the carboxyl half of POMC and revealed the presence of a new MSH-like moiety, gamma-MSH, within the 16,000-MW amino half of the precursor (16K fragment). Recent experiments have suggested that this peptide may act in synergy with ACTH to increase corticosterone and aldosterone production in vivo and in vitro. We have now isolated from a rat genomic DNA library a segment of a DNA encoding most of POMC, using as probe a mouse 144-base pair cloned cDNA fragment encoding beta-MSH and beta-endorphin. The cloned rat gene is one of two (or more) closely related POMC genes. The DNA sequence obtained shows that the cloned POMC gene is not interrupted by any intervening sequence (IVS) between the codon for amino acid 19 and the presumptive poly(A) addition site. This region of POMC encodes all the biologically active peptides mentioned above. The DNA sequence encoding the putative gamma-MSH and the coding sequence that precedes it are highly conserved between rat and cow. This may indicate an as yet unrecognized biological function(s) for the NH2-terminal portion of the 16K fragment.     

The topological configuration and conformational analysis of mRNA in translation

The theoretical model construction of mRNA hairpin structure and single-stranded structure as well as the simulation studies on RNA structure determined by the X-ray crystal diffraction and nuclear magnetic resonance revealed that in translation, after mRNA being unfolded into single-stranded structure, its topological configuration was closely correlative with the original hairpin structure. The conformational features of single-stranded mRNA appeared as helical regions alternating with curly regions to different extents, which might exert the influence on the folding of nascent polypeptide by various regulating effects including different translational rates.     

42,000-molecular weight EGF receptor has protein kinase activity

The epidermal growth factor (EGF) receptor and other growth factor receptors have been shown to possess tyrosine-specific protein kinase activity. Before the demonstration of kinase activity in growth factor receptors, tyrosine kinases of molecular weight (MW) 60,000 (60K) were found to be encoded by the src oncogene and other oncogenes related to src. Our earlier work on intracellular processing of the EGF receptor, a 170,000-MW polypeptide, provided evidence for proteolytic separation of well defined structural domains, and suggested to us the possibility of separating functional domains by limited proteolysis. The isolation of such kinase domains should facilitate comparison of the receptor/kinase with other well characterized kinases including those of oncogene origin. We report here the identification of a catalytically functional 42K kinase derived proteolytically from the isolated human EGF receptor. This fragment, comparable in size to pp60src, carries the kinase ATP-binding site, and functions catalytically even after detachment from the EGF-binding site and the major autophosphorylation region.     

The S. cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19.

Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway. In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K). In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5). Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast. Whether this activity requires additional components similar to those found in mammalian SRP is not known. We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER. Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19). We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.     

Selective effects of somatostatin-14, -25 and -28 on in vitro insulin and glucagon secretion

The widely occurring tetradecapeptide somatostatin (SRIF-14) has been variously implicated as a neurotransmitter, a neurohormone, a cybernin (local regulatory factor) and a hormone. In the first isolation of SRIF-14 from hypothalamic extracts and subsequent extracts of other tissues, peptides of higher molecular weight but with similar activity have been noted. Recently two such peptides have been characterized as the 28-amino acid SRIF-28 (from porcine gastro-intestinal tract and porcine and ovine hypothalamus and the 25-amino acid SRIF-25 (from ovine hypothalamus), each of which consists of an N-terminal extension of SRIF-14. We now report that SRIF-28 and SRIF-25 are more potent than SRIF-14 in the inhibition of insulin release, but that SRIF-14 preferentially inhibits glucagon release. This suggests that SRIF-28 and SRIF-25 are not mere biosynthetic precursors of SRIF-14 and that their differential release may be physiologically important.     

Purification and structural characterisation of human HLA-linked B-cell antigens.

The human B cell-specific alloantigen which is closely linked genetically to HLA contains two non-covalently associated, sialogycoprotein subunits of molecular weight (MW) 29,000 (p29), and 34,000 (p34). Although p29 and p34 have different amino-terminal sequences, their tyrosine peptide maps indicate considerable similarity in other portions of their polypeptide chains. Thus the genes for their proteins may have evolved by duplication of a common ancestral gene. Another lymphocyte cell surface protein of MW 16,000 (p16) has also been characterised. Both p16 and p44 (the heavy chain of HLA-A,B antigens) have been compared with p29 and p34.     

An embryo protein induced by SV40 virus transformation of mouse cells

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.     

A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing

Messenger-RNA-directed protein synthesis is accomplished by the ribosome. In eubacteria, this complex process is initiated by a specialized transfer RNA charged with formylmethionine (tRNA(fMet)). The amino-terminal formylated methionine of all bacterial nascent polypeptides blocks the reactive amino group to prevent unfavourable side-reactions and to enhance the efficiency of translation initiation. The first enzymatic factor that processes nascent chains is peptide deformylase (PDF); it removes this formyl group as polypeptides emerge from the ribosomal tunnel and before the newly synthesized proteins can adopt their native fold, which may bury the N terminus. Next, the N-terminal methionine is excised by methionine aminopeptidase. Bacterial PDFs are metalloproteases sharing a conserved N-terminal catalytic domain. All Gram-negative bacteria, including Escherichia coli, possess class-1 PDFs characterized by a carboxy-terminal alpha-helical extension. Studies focusing on PDF as a target for antibacterial drugs have not revealed the mechanism of its co-translational mode of action despite indications in early work that it co-purifies with ribosomes. Here we provide biochemical evidence that E. coli PDF interacts directly with the ribosome via its C-terminal extension. Crystallographic analysis of the complex between the ribosome-interacting helix of PDF and the ribosome at 3.7 A resolution reveals that the enzyme orients its active site towards the ribosomal tunnel exit for efficient co-translational processing of emerging nascent chains. Furthermore, we have found that the interaction of PDF with the ribosome enhances cell viability. These results provide the structural basis for understanding the coupling between protein synthesis and enzymatic processing of nascent chains, and offer insights into the interplay of PDF with the ribosome-associated chaperone trigger factor.     

Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA

The nonapeptide hormone oxytocin-like arginine-vasopressin (AVP) is synthesized as part of a larger precursor polypeptide. The precursor also includes the neurophysin molecule with which the hormone is associated in the neurosecretory granules of the hypothalamo-pituitary tract. A protein of molecular weight (Mr) approximately 20,000 has been isolated from supraoptic nuclei of rat hypothalami which, after tryptic cleavage, released a neurophysin-like molecule of Mr approximately 10,000 and an oligopeptide related to oxytocin. This result was complemented by in vitro translation of bovine hypothalamic mRNA. Among the primary translation products a single polypeptide of Mr approximately 16,500 was shown to contain antigenic determinants recognized by specific antisera against bovine neurophysin I and oxytocin. Here we report the amino acid sequence of the bovine oxytocin-neurophysin I (OT-NpI) precursor which was derived from sequence analysis of the cloned cDNA. As is the case for the bovine arginine-vasopressin-neurophysin II (AVP-NpII) precursor, the signal sequence of the OT-NpI precursor is immediately followed by the nonapeptide hormone which is connected to neurophysin I by a Gly-Lys-Arg sequence. A striking feature of the nucleic acid sequence is the 197-nucleotide long perfect homology with the AVP-NpII precursor mRNA sequence encoding the conserved middle part of neurophysins I and II.     

Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells

K1 killer toxin secreted by the K1 strain of Saccharomyces cerevisiae, has been well characterized. It is a simple protein of molecular weight (MW) 11,470 (ref. 3), encoded by a double-stranded, linear RNA plasmid, called M RNA, of MW 1.1-1.7 x 10(6) (refs 4-6). It is lethal to sensitive Saccharomyces cerevisiae which does not carry M RNA. Leakage of K+ and ATP is the first distinct response in sensitive cells, and the toxic action is thought to be due to its action as a protonophore or K+ ionophore. Recently, a further killer toxin has been found in Kluyveromyces lactis IFO 1267, and it is associated with the presence of the double-stranded linear DNA plasmids, pGK1-1 (MW 5.4 x 10(6)) and pGK1-2 (MW 8.4 x 10(6)). It has been shown, by curing pGK1-1 or deletion mapping, that the structural gene for the killer toxin and immunity-determining gene reside on the smaller plasmid. Moreover, the plasmids could be transferred from K. lactis to S. cerevisiae by protoplast fusion and protoplast transformation. As the K. lactis toxin is encoded by a DNA plasmid and has a relatively wider action spectrum than K1 killer toxin, the mode of action of the toxin is highly interesting. Here we report that K. lactis toxin inhibits adenylate cyclase in sensitive yeast cells and brings about arrest of the cells at the G1 stage.