Kurt E. Ebner

Cotranslational Membrane Insertion of the Serine Proteinase Precursor NS2B-NS3(Pro) of Dengue Virus Type 2 Is Required for Efficient in Vitro Processing and Is Mediated through the Hydrophobic Regions of NS2B

Polyprotein processing of dengue virus type 2, a positive strand RNA virus, is carried out by the host signal peptidase and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum. Using an in vitro processing system, we examined the cis andtrans cleavages of the 2B/3 and 4B/5 sites by NS2B/NS3(Pro), respectively. Lysates of BHK-21 cells coexpressing NS2B and NS3(Pro) mediated trans cleavage of the 4B/5 sitein vitro, and the protease activity was associated with the membrane fraction. To study the role of membranes in the protease activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro), and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed using a coupled in vitrotranscription/translation system (TnT). The results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner. NS2B synthesized in the presence of membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor. The cleavage products, NS2B and NS3(Pro), were membrane-associated. Furthermore, this membrane requirement was dictated by the hydrophobic regions of NS2B. Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 sitein vitro in the absence of microsomal membranes.

Isolation and structures of alligator gar (Lepisosteus spatula) insulin and pancreatic polypeptide

Insulin and a 36-residue peptide with homology to pancreatic polypeptide (PP) were isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish, by gel filtration and HPLC. Heterologous radioimmunoassays were used to detect insulin-like and PP-like immunoreactivities during purification of the two peptides. The sequence of the 36-amino acid peptide containing a C-terminal tyrosinamide was identical at 31 of 36 positions to porcine neuropeptide Y (NPY). The amino acid sequence of this peptide is YPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY-NH2. The second peptide, gar insulin, contains 52 amino acid residues and is composed of a 21-residue A chain and a 31-residue B chain. The sequence of the A chain is GIVEQCCHKPCTIYELENYCN. The sequence of the B chain is AANQHLCGSHLVEALYLVCGEKGFFYNPNKV.

Hydrophobic chromatography of galactosyltransferase

Abstract O-Glycosidic analogs of N-acetylglucosamine are good substrates for galactosyltransferase, and as the O-substituted group becomes more hydrophobic, the apparent Km decreases as much as 2000-fold. l -leucine, leucine-amide, norleucine, valine, ϵ-amino-n-caproic acid and tyrosine-agaroses all retain galactosyltransferase in the presence of 1.25 m ammonium sulfate. The enzyme is eluted quantitatively with a five- to tenfold purification by a decreasing linear gradient of ammonium sulfate. Galactosyltransferase was not specifically bound on any of a series of ω-aminoalkylagaroses tested. A simple and highly efficient procedure for the isolation of galactosyltransferase from bovine skim milk was developed and consisted of a 40–60% ammonium sulfate precipitation of the enzyme from skim milk followed by chromatography on (1) norleucine-Sepharose, (2) UDP-hexanolamine-Sepharose, and (3) α-lactalbumin-Sepharose.

The lactose synthase acceptor site: a structural map derived from acceptor studies.

SummaryA pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either α or β-, but α- is somewhat preferred. In the absence of α-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of α-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (β-configuraation). However, upon α-lactalbumin binding the aglycone specificity allows for axial (α-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that α-lactalbumin binds to a hydrophobic site some distance from the C-2 group.

Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: amino acid sequences of oxyntomodulin and glucagon-like peptide

Oxyntomodulin, glucagon, and a glucagon-like peptide (GLP) have been isolated from the endocrine pancreas of the alligator gar (Lepisosteus spatula), a ganoid fish. The three peptides were isolated by gel filtration and HPLC and were identified by size, composition, and glucagon-like immunoreactivity. The amino acid sequences of the oxyntomodulin and GLP were determined. The oxyntomodulin contains 36 amino acid residues and its sequence is H S Q G T F T N D Y S K Y L D T R R A Q D F V Q W L M S T K R S G G I T. The composition of the glucagon is identical to the N-terminal 29 residues of the gar oxyntomodulin. The single form of GLP found contains 34 amino acid residues in the following sequence: H A D G T Y T S D V S S Y L Q D Q A A K K F V T W L K Q G Q D R R E. These findings suggest that all three peptides are derived from a common precursor.

Isolation and characterization of rat α-Lactalbumin: A glycoprotein

Abstract α-Lactalbumin was purified to homogeneity from rat milk. Rat α-lactalbumin, in contrast to other α-lactalbumins, is a glycoprotein and exhibits an abnormally high molecular weight when obtained by gel filtration or electrophoresis in sodium dodecyl sulfate. The molecular weight by sedimentation equilibrium is 15 400 ± 5% and of the reduced and alkylated protein is 16 000 when determined by thin-layer chromatography in 6 M guanidine hydrochloride. At least, three major charge forms, all containing carbohydrate and active in the lactose synthetase reaction were demonstrated. The amino acid composition reveals a high proline content which is reflected in a low α-helical content.

Activity of alkylated prolactin and human growth hormone in receptor and cell assays

The disulfide bonds of two lactogenic hormones, ovine prolactin (oPRL) and human growth hormone (hGH), were reduced with dithiothreitol under denaturing conditions and alkylated with iodoacetic acid. The modified hormones were assayed for their ability to bind the plasma membrane-bound receptor for lactogenic hormone found in the rabbit mammary gland. S-Carboxymethylated ovine prolactin (SCM-oPRL) with all six cysteine residues modified had a nearly 300-fold decrease in binding as compared to native oPRL in a competitive binding assay using [125I]ovine prolactin. The S-carboxymethylated human growth hormone (SCM-hGH) had all four of its cysteine residues modified. It showed only a slightly reduced ability to bind the rabbit mammary gland prolactin receptor in a competitive binding assay with [125I]ovine prolactin. The two modified hormones were assayed for their ability to stimulate proliferation of the lactogen-dependent Nb 2 lymphoma cell line. SCM-oPRL required concentrations greater than 1 X 10(5) that of native oPRL to stimulate 50% of the maximum cell growth. SCM-hGH retained a significant amount of its ability to stimulate the Nb 2 lymphoma cells.

pH dependence of dissociation of the ovine prolactin rabbit mammary receptor complex

Ovine prolactin specifically bound to rabbit mammary membrane prolactin receptor was rapidly dissociated in a pH dependent manner with 0.1M ammonium acetate. Up to 75% of the bound hormone was dissociated at pH 4.0 or lower in less than 5 minutes. The pK of the dissociation was 4.7, implicating one or more critical carboxyl groups. Exposure of the membrane bound receptor to the dissociating buffer for up to one hour did not reduce its ability to bind hormone. The dissociated hormone was characterized as intact ovine prolactin by Bio-Gel P-150 gel chromatography and by its ability to bind to fresh rabbit prolactin receptor with the same binding affinity as native hormone.

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

Abstract An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD + as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD + using 2-mercaptoethanol as the substrate. Although the K m for mercaptoethanol is much lower than that for ethanol, 30 μ m as compared to 0.625 m m , the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K i for 2-mercaptoethanol is 14 μ m . 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.

A radioimmunoassay for mouse alpha-lactalbumin.

alpha-Lactalbumin has been purified from mouse milk by the use of ammonium sulfate precipitation, Bio-Gel P-100 chromatography and DEAE-cellulose chromatography. Mouse alpha-lactalbumin exists as two major charge forms with the same molecular weight. Both charge forms have been used in the development of a radioimmunoassay to mouse alpha-lactalbumin. The assay is specific for mouse alpha-lactalbumin, reacting identically with both forms, and sensitive with a minimal detectable concentration of 0.35 ng. alpha-Lactalbumin induced in mouse mammary gland explants has been measured with the use of the radioimmunoassay and is detectable in both the explants and media of the cultures.