Corné H. W. Klaassen

Functional expression of gastric H,K-ATPase using the baculovirus expression system

A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α‐ as well as the β‐subunit of the gastric H.K‐ATPase. Sf9 cells infected with this virus exhibited a K+‐ and SCH 28080‐sensitive ATP‐dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K‐ATPase. This activity was not present in control membranes containing only one of the two H,K‐ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K‐ATPase.

The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity.

Recombinant human purple acid phosphatase (recHPAP) provides a convenient experimental system for assessing the relationship between molecular structure and enzymatic activity in mammalian purple acid phosphatases (PAPs). recHPAP is a monomeric protein with properties similar to those of uteroferrin (Uf) and other PAPs isolated as single polypeptide chains, but its properties differ significantly from those of bovine spleen PAP (BSPAP) and other PAPs isolated as proteolytically “clipped” forms. Incubation of recHPAP with trypsin results in proteolytic cleavage in an exposed region near the active site. The product is a tightly associated two‐subunit protein whose collective spectroscopic and kinetics properties resemble those of BSPAP. These results demonstrate that the differences in spectroscopic and kinetics properties previously reported for mammalian PAPs are the result of proteolytic cleavage. Mass spectrometry shows that a three‐residue segment, D‐V‐K, within the loop region is excised by trypsin. This finding suggests that important interactions between residues in the excised loop and one or more of the groups that participate in catalysis are lost or altered upon proteolytic cleavage. Analysis of available structural data indicates that the most important such interaction is that between Asp 146 in the exposed loop and active‐site residues Asn 91 and His 92. Loss of this interaction should result in both an increase in the Lewis acidity of the FeII ion and an increase in the nucleophilicity of the FeIII‐bound hydroxide ion. Proteolytic cleavage thus constitutes a potential physiological mechanism for regulating the activity of PAP in vivo.

Large‐scale purification of functional recombinant human aquaporin‐2

The homotetrameric aquaporin‐2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre‐eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his‐tagged human AQP2 (HT‐AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT‐AQP2 could be obtained per liter of bioreactor culture. HT‐AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93±0.03×10−13 cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large‐scale expression of functional recombinant human AQP2 that is suitable for structural studies.

Tyrosine Structural Changes Detected during the Photoactivation of Rhodopsin

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho → MII difference spectra were obtained at 10 °C for unlabeled recombinant Rho and isotope-labeledl-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enrichedl-[ring-2H4]Tyr and isolated using a His6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR → M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the F-helix, may form a hinge that facilitates α-helix movement.

Gastric H+/K+-ATPase

The gastric H+/K+-ATPase is an enzyme located in the tubu-lovesicular system and the apical membrane of the parietal cell and plays a crucial role in the process of gastric acid secretion. The enzyme catalyses an electroneutral exchange of H+ versus K+. It consists of a catalytic α-subunit and a heavily glycosylated β-subunit, both of which are rather homologous to their counterparts in Na+/K+-ATPase. In this review, molecular and genetic aspects of this enzyme are reviewed with reference to similar enzymes in other tissues.

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1 Rat histamine H2 receptors were epitope‐tagged with six histidine residues at the C‐terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope‐tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2 The His‐tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]‐aminopotentidine (APT) binding studies. 3 In Sf9 cells expressing the His‐tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3). 4 The expression of the His‐tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg−1 protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×106 copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol‐β‐cyclodextrin complex resulted in an increase of [125I]‐APT binding up to 169±5% (mean±s.e.mean, n=3). 5 The addition of 0.03 mM cholesterol‐β‐cyclodextrin complex did not affect histamine‐induced cyclic AMP production. The EC50 value of histamine was 3.1±1.7 μM in the absence of cholesterol‐β‐cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol‐β‐cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/106 cells in the absence and 81±11 pmol/106 cells in the presence of 0.03 mM cholesterol‐β‐cyclodextrin complex (mean±s.e.mean, n=3). 6 Immunofluorescence studies with an antibody against the His‐tag revealed that the majority of the His‐tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7 These experiments demonstrate the successful expression of His‐tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His‐tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.

Evaluation of the trophic effect of longterm treatment with the histamine H2 receptor antagonist loxtidine on rat oxyntic mucosa by differential counting of dispersed cells.

To evaluate whether the general trophic effect of gastrin on the oxyntic mucosa is an indirect effect mediated by histamine H2 receptors, sustained 24 hour hypergastrinaemia was induced in Sprague-Dawley rats by treatment with the long acting and potent histamine H2 antagonist loxtidine for five months. The trophic effect was assessed by weight, enumeration of total mucosal cells, parietal cells, and enterochromaffin like cells in smears stained for the actual cells after enzymatic dispersion of the mucosa, and by biochemical analysis of oxyntic mucosal homogenates. The weight of the whole stomach and the oxyntic mucosa increased by 12.7% (p = 0.016) and 27.5% (p = 0.006), respectively. Total oxyntic mucosal protein content increased by 28.7% (p = 0.058). Total numbers of mucosal cells and parietal cells increased by 11.9% (NS) and 24.1% (NS), respectively. The amount of the parietal cell specific enzyme H+,K(+)-ATPase was unchanged. On the other hand, the number of enterochromaffin like cells and related parameters, histidine decarboxylase activity and histamine content of the oxyntic mucosa, showed a pronounced and significant increase. It is concluded that the general trophic effect of gastrin on the oxyntic mucosa is not mediated by the histamine H2 receptor. The tropic effect of gastrin on the parietal cell seems, in contrast with that on the enterochromaffin like cell, not to be specific but only reflecting the general trophic effect on the oxyntic mucosa.

The K+ affinity of gastric H+,K+-ATPase is affected by both lipid composition and the β-subunit

It is generally assumed that negatively charged residues present in the alpha-subunit of gastric H(+),K(+)-ATPase are involved in K(+) binding and transport. Despite the fact that there is no difference between various species regarding these negatively charged residues, it was observed that the apparent K(+) affinity of the pig enzyme was much lower than that of the rat H(+),K(+)-ATPase. By determining the K(+)-stimulated dephosphorylation reaction of the phosphorylated intermediate K(0.5) values for K(+) of 0.12+/-0.01 and 1.73+/-0.03 mM were obtained (ratio 14.4) for the rat and the pig enzyme, respectively. To investigate the reason for the observed difference in K(+) sensitivity, both enzymes originating from the gastric mucosa were either reconstituted in a similar lipid environment or expressed in Sf9 cells. After reconstitution in K(+)-permeable phosphatidylcholine/cholesterol liposomes K(0.5) values for K(+) of 0.16+/-0.01 and 0.35+/-0.05 mM for the rat and pig enzyme respectively were measured (ratio 2.2). After expression in Sf9 cells the pig gastric H(+),K(+)-ATPase still showed a 4.1 times lower K(+) sensitivity than that of the rat enzyme. This means that the difference in K(+) sensitivity of the rat and pig gastric H(+), K(+)-ATPase is not only due to a different lipid composition but also to the structure of either the alpha- or beta-subunit. Expression of hybrid enzymes in Sf9 cells showed that the difference in K(+) sensitivity between the rat and pig gastric H(+),K(+)-ATPase is primarily due to differences in the beta-subunit.

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (Ki=150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.

Functional Expression of Gastric H,K-ATPase in Sf9 Cells using the Baculovirus Expression System

The mechanism of action of H,K-ATPase is still only partly understood. Site-directed mutagenesis and chimere production might give useful tools to enlarge our insight in structure-function relationship of this enzyme. Functional expression in vitro is a prerequisite for such studies. The baculovirus expression system makes advantage of the high level expression of certain viral proteins not essential for viral replication in a cell line from the fall armyworm Spodoptera frugiperda (Sf9 cells) [Vlak and Keus,1990]. Two such proteins are polyhedrin and p10 protein. Both proteins can reach expression levels of more than 30% in Sf9 cells, dependent on the stage of infection. Replacing polyhedrin or p10 coding sequences by homologous recombination between wild-type viral DNA and a transfer vector DNA has led to the production of recombinant viruses expressing large amounts of recombinant proteins. Many complex membrane proteins as well as cytosolic proteins have successfully been expressed using this system [Parker et al.,1991; Janssen et al.,1991; Fafournoux et al.,1991; Li et al.,1992; Smith et al.,1992; De Tomaso et al.,1993]. In order to produce multiple subunit proteins, simultaneous expression of two or more proteins has been made possible by using either one transfer vector containing two or more promoters [Belyaev and Roy,1993] or by co-infection of Sf9 cells using a mixture of two or more recombinant baculoviruses. We therefore decided to study the in vitro synthesis of H,K-ATPase subunits using the baculovirus expression system.