Isolation and characterization of human platelet glycoprotein IX

Human platelet glycoprotein IX is a small (Mr 17,000) surface glycoprotein present in the plasma membrane as a 1:1 non-covalent complex with glycoprotein Ib (Mr 165,000). Glycoprotein IX was purified to near homogeneity by sequential wheat germ agglutinin and immuno-affinity chromatography, followed by gel filtration chromatography under denaturing conditions. Purified glycoprotein IX was characterized by determination of its amino acid composition and its NH2-terminal amino acid sequence (thr-lys-asp-xxx-pro-ser-xxx-leu-thr-(thr)2-arg-ala-(leu)-(glu)-xxx-met- gly), and monospecific anti-glycoprotein IX antibody was prepared. The study outlines a useful approach for separating and characterizing glycoprotein IX, free from other members of the glycoprotein Ib complex.     

Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11beta-hydroxysteroid dehydrogenase and the 50-kDa esterase.

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11beta-hydroxysteroid dehydrogenase, isozyme 1 (11beta-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11beta-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy and electron microscopic immunogold labeling. The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membranes in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl group and N-terminal 30-residue membrane anchor. The orientation of the cytochrome b5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.     

Decreased Silica Land–sea Fluxes through Damming in the Baltic Sea Catchment – Significance of Particle Trapping and Hydrological Alterations

We tested the hypothesis that reservoirs with low water residence time and autochthonous production influence river biogeochemistry in eutrophied river systems draining cultivated watersheds. The effect of a single artificial water reservoir and consecutive reservoirs on silica (Si) river fluxes is exemplified by the moderately dammed Vistula River and the heavily regulated Daugava River that are compared with the practically undammed Oder River. The sum of the discharge weighted annual mean biogenic silica (BSi) and dissolved silicate (DSi) concentrations in the rivers Oder, Vistula and Daugava were about 160 μ M (40 + 120 μ M), 150 μ M (20 + 130 μ M) and 88 μ M (6 + 82 μ M), respectively. Assuming BSi and DSi concentrations as observed in the Oder River as typical for eutrophied but undammed rivers, complete trapping of this BSi could have lowered Si fluxes to the Baltic Sea from rivers with cultivated watersheds by 25%. The superimposed effect of hydrological alterations on reduced Si land–sea fluxes is demonstrated by studies in the boreal/subarctic and oligotrophic rivers Kalixälven and Lueälven. The DSi yield of the heavily dammed Luleälven (793 kg km^?2 yr^?1) constituted only 63% of that was found in the unregulated Kalixälven (1261 kg km^?2 yr^?1), despite the specific runoff of the Luleälven (672 mm m^?2 yr^?1) being 19% higher than that of theKalixälven (563 mm m^?2 yr^?1); runoff normalized DSi yield of the former, regulated watershed, was only half the DSi yield of the latter, unperturbed watershed. Based on these findings, it is hypothesized here that perturbed surface water–groundwater interactions are the major reasons for the reduced annual fluctuations in DSi concentrations as also seen in the heavily dammed and eutrophic river systems such as the Daugava and Danube.     

The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450

The complete covalent structure of the constitutive cytochrome P-450, form 3b, from rabbit liver microsomes was determined. The apocytochrome contains 490 amino acid residues in a single polypeptide chain, Mr = 55,860. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. The constitutive form is only 46% homologous to the phenobarbital-induced cytochrome P-450 (Heinemann, F. S., and Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201) and contains a deletion at position 22. Strongly conserved regions include Cys435 and a previously identified tryptic peptide, residues 345-357. The distribution of hydrophobic segments is used to predict the membrane topology of the protein, and four possible orientations of this protein in the membrane are presented.     

Characterization of the covalent cross-links of the active sites of amidinated cytochrome b5 and NADH:cytochrome b5 reductase

Preparations of amidinated cytochrome b5 and cytochrome b5 reductase, cross-linked by using a soluble carbodiimide to promote the formation of covalent bonds between carboxyl groups of the hemeprotein and nucleophilic residues of the flavoprotein at the surfaces involved in protein-protein contacts during electron transfer, have been used to characterize the charge pair interactions that occur during electron transfer between the free proteins. Sequence analyses of tryptic, V8 protease-, and Asp-N protease-generated peptides show that the heme propionyl carboxyl group at the surface of the cytochrome forms an ester bond with Ser162 of the reductase, thus implicating Lys163 as the normal participant in ionic bonding between the active sites of the two proteins. Moreover, Lys41 and Lys125 directly form amide bonds with carboxyl residues on the active-site surface of the cytochrome. In the case of Lys41, this involves Glu52 and/or Glu60, and Glu47 and/or Glu48 for Lys125, again implicating these residues as the groups that form charge pairs during normal interactions between the active sites of the two proteins.     

Covalent structure of the membranous segment of horse cytochrome b5. Chemical cleavage of the native hemoprotein

The complete covalent structure of the membranous segment of horse liver cytochrome b5 has been determined. This peptide spans residues 91 to 133 of the cytochrome molecule, and contains the segment responsible for the association of the hemoprotein with microsomal or synthetic vesicles. Two peptides, residues 91 to 127 and 128 to 133, comprising the entire membranous moiety were isolated from a tryptic digest of urea-denatured apoprotein. The membranous segment (residues 91 to 127) could be separated from all other tryptic peptides by a single gel filtration step. Trypsin digestion of succinylated cytochrome produced similar peptides, residues 89 to 127 and 128 to 133. The covalent structures for residues 89 to 127 and 128 to 133 were derived from automated sequenator analysis of tryptic peptides. Chemical cleavage at tryptophanyl, or methionyl residues, or both, by the method of Ozols and Gerard ((1977) J. Biol. Chem. 252, 5986-5989) provided the overlapping peptides from which the following unique sequence was deduced: (formula: see text).     

Gastric inhibitory polypeptide does not inhibit gastric emptying in humans

The insulinotropic gut hormone gastric inhibitory polypeptide (GIP) has been demonstrated to inhibit gastric acid secretion and was proposed to possess "enterogastrone" activity. GIP effects on gastric emptying have not yet been studied. Fifteen healthy male volunteers (23.9 +/- 3.3 yr, body mass index 23.7 +/- 2.3 kg/m(2)) were studied with the intravenous infusion of GIP (2 pmol.kg(-1).min(-1)) or placebo, each administered to the volunteers on separate occasions from -30 to 360 min in the fasting state. At 0 min, a solid test meal (250 kcal containing [(13)C]sodium octanoate) was served. Gastric emptying was calculated from the (13)CO(2) exhalation rates in breath samples collected over 360 min. Venous blood was drawn in 30-min intervals for the determination of glucose, insulin, C-peptide, and GIP (total and intact). Statistical calculations were made by use of repeated-measures ANOVA and one-way ANOVA. During the infusion, GIP rose to steady-state concentrations of 159 +/- 15 pmol/l for total and 34 +/- 4 pmol/l for intact GIP (P < 0.0001). Meal ingestion further increased GIP concentrations in both groups, reaching peak levels of 265 +/- 20 and 82 +/- 9 pmol/l for total and 67 +/- 7 and 31 +/- 9 pmol/l for intact GIP during the administration of GIP and placebo, respectively (P < 0.0001). There were no differences in glucose, insulin, and C-peptide between the experiments with the infusion of GIP or placebo. Gastric half-emptying times were 120 +/- 9 and 120 +/- 18 min (P = 1.0, with GIP and placebo, respectively). The time pattern of gastric emptying was similar in the two groups (P = 0.98). Endogenous GIP secretion, as derived from the incremental area under the curve of plasma GIP concentrations in the placebo experiments, did not correlate to gastric half-emptying times (r(2) = 0.15, P = 0.15 for intact GIP; r(2) = 0.21, P = 0.086 for total GIP). We conclude that gastric emptying does not appear to be influenced by GIP. The secretion of GIP after meal ingestion is not suppressed by its exogenous administration. The lack of effect of GIP on gastric emptying underlines the differences between GIP and the second incretin glucagon-like peptide 1.     

Model‐based optimization and pO2 control of fed‐batch Escherichia coli and Saccharomyces cerevisiae cultivation processes

A model‐based approach for optimization and cascade control of dissolved oxygen partial pressure (pO₂) and maximization of biomass in fed‐batch cultivations is presented. The procedure is based on the off‐line model‐based optimization of the optimal feeding rate profiles and the subsequent automatic pO₂ control using a proposed cascade control technique. During the model‐based optimization of the process, feeding rate profiles are optimized with respect to the imposed technological constraints (initial and maximal cultivation volume, cultivation time, feeding rate range, maximal oxygen transfer rate and pO₂ level). The cascade pO₂ control is implemented using activation of cascades for agitation, oxygen enrichment, and correction of the preoptimized feeding rate profiles. The proposed approach is investigated in two typical fed‐batch processes with Escherichia coli and Saccharomyces cerevisiae. The obtained results show that it was possible to achieve sufficiently high biomass levels with respect to the given technological constraints and to improve controllability of the investigated processes.     

SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.