Bacterial synthesis of recombinant alpha-human atrial natriuretic polypeptide

The high-level synthesis of alpha-human atrial natriuretic polypeptide hormone in Escherichia coli has been achieved based on the idea that the yield of a small, basic and unstable polypeptide, such as the natriuretic polypeptide, would be improved by fusion with an appropriate protective polypeptide to construct a neutral fused polypeptide. We prepared an expression vector, pCLaHtrp3t, coding a neutral polypeptide containing 130 amino acid residues in which the polypeptide hormone was fused to a newly designed protective polypeptide through lysine as an enzymatically cleavable residue. The fused polypeptide was synthesized at the high level of 32% of total cellular proteins and at 4.7 X 10(6) molecules per single cell. It was recovered as cellular insoluble fraction and purified to homogeneity. For the isolation of the peptide hormone from the resultant fused polypeptide, Achromobacter protease I, a lysine-specific endopeptidase was used, because it has sufficient activity even in 8 M urea. The recombinant natriuretic polypeptide was indistinguishable from native alpha-human atrial natriuretic polypeptide as regards amino acid sequence as well as biological activity.     

Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus.

The light-harvesting complex I (LHI) of Rhodobacter capsulatus is an oligomer of basic subunits each consisting of the two different pigment-binding polypeptides LHI alpha and LHI beta, encoded by the pufA (LHI alpha) and pufB (LHI beta) genes. Pulse-labeling experiments showed that in the presence of the LHI alpha polypeptide, the LHI beta polypeptide was inserted earlier into the intracytoplasmic membrane than was the LHI alpha polypeptide. Each of the pufA and pufB genes was deleted to test whether the LHI alpha and beta polypeptides, respectively, are inserted into the intracytoplasmic membrane independently of the LHI partner polypeptide. Neither deletion mutant strain formed the LHI antenna, but a functional reaction center complex was present. Pulse-labeling experiments indicated that the LHI beta polypeptide was inserted into the intracytoplasmic membrane with the same kinetics and in the same amounts regardless of whether the LHI alpha polypeptide was present. However, the LHI beta polypeptide did not accumulate in the membrane in the absence of the LHI alpha protein but was degraded linearly within about 12 min. In contrast to the LHI beta protein, only trace amounts of the LHI alpha polypeptide were inserted into or attached to the membrane if the LHI beta polypeptide was not synthesized.     

Amino acid sequences of the aplpha and beta chains of adult hemoglobin of the tupai, Tupaia glis.

Globin prepared from hemoglobin of adult tupai (Tupaia glis) was separated into alpha and beta polypeptide chains by CM-cellulose column chromatography. The S-aminoethylated alpha polypeptide chain and S-carboxymethylated beta polypeptide chain were each digested with trypsin, and the sequences of all the peptides thus obtained were established. The ordering of these tryptic peptides in the alpha and beta polypeptide chains was deduced from the homology of their primary structures with that of human adult hemoglobin. In this way the primary structures of the alpha and beta polypeptide chains of tupai hemoglobin were established; 27 amino acids in the alpha polypeptide chain and 26 in the beta chain differ from those in human adult hemoglobin.     

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.     

Protein composition of Rhodopseudomonas sphaeroides outer membrane.

The outer membrane polypeptide profile of Rhodopseudmonas sphaeroides was characterized. Solubilization of the outer membrane at 75 or 100 degrees C as opposed to room temperature resulted in the dissociation of 75-, 72-, and 68-kilodalton (kdal) polypeptide aggregates into 29-, 26.5-, and 21.5-kdal polypeptides, respectively, and a shared 47-kdal subunit. Similarly, an 88.5-kdal polypeptide dissociates into a 45-kdal monomeric form, and the electrophoretic mobility of a 58.5-kdal polypeptide was altered to 83 kdal.Lysozyme treatment of outer membrane fractions altered the 21.5-kdal polypeptide mobility to 23 kdal. The presence of lipid in both the 47-kdal polypeptide and an 8- to 10-kdal polypeptide was demonstrated by lipid staining and [14C]acetate incorporation. The lipid component of the 47-kdal polypeptide was neither lipopolysaccharide nor phospholipid. The 8- to 10-kdal polypeptide may be the equivalent of the Braun lipoprotein. Outer membrane fractions isolated from R. sphaeroides-specific phage RS1-resistant mutants were deficient in several of the high-molecular-weight aggregates involving the 47-kdal polypeptide.     

Polypeptide analysis of genotypic variants of occluded Heliothis spp. baculoviruses

The structural polypeptides of 12 baculovirus isolates which included nuclear polyhedrosis viruses (NPVs) and granulosis viruses (GVs) obtained from four different species of the insect genus Heliothis collected in different geographical regions of the world were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The matrix proteins were compared according to their molecular weights and peptide profiles produced after limited proteolysis. Examination of the matrix and virion polypeptide profiles revealed three major polypeptide phenotypes which corresponded to the three baculovirus morphological groups; singly embedded nuclear polyhedrosis viruses (SNPVs), multiply embedded nuclear polyhedrosis viruses (MNPVs), and granulosis viruses (GVs). Enveloped nucleocapsid polypeptide profiles of isolates within each NPV phenotype differed in only one polypeptide whereas the two GV isolates differed by as many as five polypeptides. Nucleocapsid polypeptide profiles of isolates within each of the NPV subgroups were identical while those profiles from the GV nucleocapsids differed slightly in molecular weight of one polypeptide.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

Synthesis of DNA polymerase by in vitro translation of calf RNA

Synthesis of alpha-polymerase in translation mixtures containing calf thymus poly(A+) RNA was examined by activity gel analysis and by immuno-binding with a monoclonal antibody to calf thymus alpha-polymerase. Activity gel analysis indicated that a DNA polymerase catalytic polypeptide of Mr = approximately 120,000 had been synthesized. Immunobinding experiments indicated that an immunoreactive polypeptide of about the same size had been formed in vitro. Sucrose gradient centrifugation of calf thymus total RNA revealed that mRNA encoding the approximately 120,000-Mr DNA polymerase polypeptide sedimented at about 16S. This approximately 120,000-Mr catalytic polypeptide corresponds in size to an alpha-polymerase catalytic polypeptide found earlier in crude extracts of calf cells.     

Interactions of helical polypepetide segments which span the hydrocarbon region of lipid bilayers. Studies of the gramicidin A lipid-water system.

The polypeptide gramicidin A in a dimeric form is considered to form a helical structure which spans the hydrocarbon region of lipid bilayers. In the present investigation it is used as a model for the interactions of the polypeptide segments of transmembrane proteins within the hydrocarbon region of the lipid bilayers of biomembrane structures. A variety of physical techniques (X-ray diffraction, differential scanning calorimetry, optical and electron microscopy, Raman and electron spin resonance spectroscopy) are applied to a study of the interactions of this polypeptide within the phospholipid bilayers of dimyristoyl and dipalmitoyl lecithins in water, at temperatures both above and below the main endothermic phase transition of the pure lipids.Above the transition temperature of the lipid, the Raman studies show that the polypeptide perturbs the fluid lipid environment and causes a marked decrease in the number of gauche isomers of the lipid hydrocarbon chains, even at quite low relative molar concentrations of the polypeptide to lipid (1:150). At concentrations of phospholipid to polypeptide of less than 5:1, the electron spin resonance studies show the existence of two lipid regions within the bilayer. One region corresponds to the relatively fluid lipid region normally observed at these temperatures and the other to a relatively rigid lipid region. The latter is considered to arise from clusters of the polypeptide in which some of the lipid is entrapped.Below the lipid phase transition temperature, the pretransition endotherm observed with pure lipid-water systems is removed by small molar concentrations of the polypeptide (1:50) and the rippled appearance observed in freeze-fracture electron micrographs with pure dimyristoyl lecithin-water dispersions is replaced by a smooth appearance.The main lipid phase transition becomes broadened by the presence of increasing amounts of the polypeptide within the lipid bilayer as indicated by calorimetry, and electron spin resonance spectroscopy. The enthalpy of the lipid transition decreases linearly with increasing amounts of the polypeptide until, with dipalmitoyl lecithin, a concentration of approximately 20 lipids per polypeptide is reached. This is considered to correspond to the onset of an aggregation process which produces localised polypeptide-lipid clusters within the plane of the membrane.At concentrations of polypeptide less than five lipids per polypeptide, freezefracture electron microscopy shows the presence of liposomes with smooth fracture faces. At higher polypeptide concentrations, sheet-like structures are observed with smooth fracture faces.When a mixed lipid-water system (dilauroyl and dipalmitoyl lecithin) containing low concentrations of the polypeptide is slowly cooled, the calorimetric evidence shows that the polypeptide moves preferentially into the lower melting region of the bilayer, whereas at higher polypcptide eoncentrations a mixing of the two lipids takes place.The various results are discussed to provide insight pertinent to the organisation, interactions, aggregation properties, boundary layer and packing arrangements of helical polypeptides and proteins in reconstituted systems and natural biomembranes.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 4. Genetic polymorphism of cytosol 100k polypeptide

Summary We describe a genetic polymorphism of a human cellular polypeptide with mol. wt. 100,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, and 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is one of the abundant polypeptides of B-lymphoblastoid cells, T-lymphoblastoid cells, fibroblasts, and HeLa cells. The data indicate that the cytosol polypeptide with mol.wt. 100,000 shows a genetic polymorphism determined by aew autosomal locus. It is proposed that the polypeptide and its locus be temporarily designated cytosol 100k polypeptide (C100k polypeptide) and C100P, respectively. In a Japanese population, the gene frequencies of C100P ¹ and C100P ² were 0.907 and 0.093, respectively.     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis

Summary We describe a genetic polymorphism of cytosol polypeptide with mol.wt. of 20,000 detected in lymphocytes the arythrocytes by two-dimensional gel electrophoresis. Three different electrophoretic phenotypes (type 1-1, 2-1, and 2-2) of the polypeptide have been identified in a Japanese population. Family studies indicate that the phenotypes are determined by two common alleles at a single autosomal locus. The polypeptide is present in the cytosol of various kinds of cells and is abundant in erythrocytes. The data on a gel filtration of the erythrocyte cytosol proteins on a Sephadex G-100 column suggest that the polypeptide exists as a dimer in cells. In nine out of 79 individuals, the phenotypes of the polypeptide were different from those of glyoxalase 1 (GLO1) which has similar properties in subunit size, cell distribution, and allele frequencies. These date indicate that the polypeptide with mol. wt. of 20,000 is a new polymorphic cellular polypeptide. We propose that the polypeptide be temporarily designated as cytosol polypeptide with mol. wt. of 20,000 (CP20) and that the gene for CP20 be designated as CP20. The gene frequencies of two common alleles (CP20 ¹ and CP20 ²) are 0.955 and 0.045, respectively, in a Japanese population.     

Immunochemical analysis of molecular forms of mammalian DNA ligases I and II

Using specific antibodies against calf thymus DNA ligases I and II (EC 6.5.1.1), we have investigated the polypeptide structures of DNA ligases I and II present in the impure enzyme preparations, and estimated the polypeptides of DNA ligases I and II present in vivo. Immunoblot analysis of DNA ligase I after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 130-kDa polypeptide as a major one in the enzyme preparations from calf thymus throughout the purification. In addition to the 130-kDa polypeptide, a 200-kDa polypeptide was detected in the enzyme preparations at the earlier steps of the purification, and a 90-kDa polypeptide was observed as a minor one in the enzyme preparations at the later steps of the purification. The polypeptides with molecular weight of 130 000 and 90 000 were detected by SDS-polyacrylamide gel electrophoresis of DNA ligase I-[3H]AMP complex. These results suggest that a 200-kDa polypeptide of DNA ligase I present in vivo is degraded to a 130-kDa polypeptide and then to a 90-kDa polypeptide during the isolation and purification procedures. On the other hand, the monospecific antibody against calf thymus DNA ligase II cross-reacted with only a 68 kDa polypeptide in the enzyme preparations throughout the purification, suggesting that the 68-kDa polypeptide is a single form of calf thymus DNA ligase II present in vivo as well as in vitro.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

Origin of the gamma polypeptide of the Na+/K+-ATPase

The Na+/K+-ATPase purified from lamb kidney contains a gamma polypeptide fraction which is a collection of fragments derived from the alpha and beta polypeptides of the enzyme. This fraction has the solubility characteristics of a proteolipid and was isolated either by high performance liquid chromatography (size exclusion chromatography) in 1% sodium dodecyl sulfate or by sequential organic extraction of purified lamb kidney Na+/K+-ATPase. Formation of gamma polypeptide(s) from detergent solubilized holoenzyme was accelerated by sulfhydryl containing reagents and was unaffected by addition of inhibitors of proteolytic enzymes. Treatment of the holoenzyme with the photoaffinity reagent N-(2-nitro-4-azidophenyl)[3H]ouabain ([3H]NAP-ouabain) labeled the alpha polypeptide and the gamma polypeptide fraction but not the beta polypeptide. Amino acid sequence analysis of one gamma polypeptide preparation revealed homology of one component of this fraction with the N-terminus of the beta subunit of the Na+/K+-ATPase. Amino acid analysis of two preparations of proteolipid showed similar amino acid compositions with a peptide derived from the alpha subunit. The insolubility and complexity of the gamma polypeptide(s)/proteolipid fraction appears to preclude a conclusive sequence analysis of all components of this fraction.     

Novel Regulatory Role of Phosphorylated Clathrin Light Chain β in Bovine Brain Coated Vesicles

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50-kDa polypeptide in CCVs with phosphorylated LC beta results in a rapid dephosphorylation of the 50-kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LC beta prevent the modulatory effect of phosphorylated LC beta on the 50-kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LC beta has a modulatory role in CCVs. The data also suggest that phosphorylated LC beta promotes activation of a coated vesicle phosphatase.     

Effect of Jasmonic, Salicylic, and Abscisic Acids on [14C]Leucine Incorporation into Proteins of Pea Leaves

All investigated exogenous phytohormones (jasmonic, salicylic, and abscisic acids) induced the appearance of ¹⁴C-label in a polypeptide with molecular mass 29 kD that was not found in the control; these acids also increased [¹⁴C]leucine incorporation into a 25-kD polypeptide and decreased such incorporation into a 45-kD polypeptide. This can be considered as a nonspecific response of the plants to the action of these hormones. Salicylic and abscisic (but not jasmonic) acids induced the synthesis of a 19-kD polypeptide, and jasmonate induced the synthesis of a 96-kD polypeptide.     

The ability of pancreatic polypeptides (APP and BPP) to return to normal the hyperglycaemia, hyperinsulinaemia and weight gain of New Zealand obese mice.

Intraperitoneal injections of avian pancreatic polypeptide (APP) and bovine pancreatic polypeptide (BPP) are capable of returning to normal the hyperinsulinaemia, hyperglycaemia and weight gain of New Zealand obese mice. The lag glucose tolerance also becomes indistinguishable from normal. The mechanism whereby these polypeptides cause reversion is not known. Reversion can also be brought about by the intraperitoneal implantation of islets from white mice into New Zealand obese animals. The implanted islets secrete mouse pancreatic polypeptide. We conclude that the New Zealand obese syndrome arises from a genetic lack of mouse pancreatic polypeptide. We suggest that in humans a lack of pancreatic polypeptide might manifest as a syndrome analogous to that found in New Zealand obese mice.     

Human pancreatic polypeptide has a marked diurnal rhythm that is affected by ageing and is associated with the gastric TFF2 circadian rhythm

Normal circadian variations in vasoactive intestinal polypeptide, somatostatin, cholecystokinin and pancreatic polypeptide were measured to determine if these alter with aging and to identify gastrointestinal regulatory hormones that might control the dramatic diurnal variation in the gastric cytoprotective trefoil protein TFF2. Plasma pancreatic polypeptide concentrations showed a marked diurnal rhythm (p < 0.0001). Basal and postprandial pancreatic polypeptide concentrations increased with age (p < 0.01). The timing of the diurnal rhythm was consistent with pancreatic polypeptide inhibiting TFF2 secretion and there was a negative association between pancreatic polypeptide and TFF2 concentrations (p < 0.002). The much higher pancreatic polypeptide concentrations in older people will induce increased satiety that may contribute to 'anorexia of ageing'. These results identify potential therapies for treatment of gastric mucosal morbidity and age-associated loss of appetite.