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C H Klaassen

Publications and source records attributed to C H Klaassen.

At least 19 recordsLinked to original sources
PubMed1998

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 degrees C for unlabeled recombinant Rho and isotope-labeled L-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[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 alpha-helix movement.

Amino Acid Sequence↗
PubMed1998

Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates.

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

Animals↗
PubMed1997

Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.

Animals↗
PubMed1997

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 microM) 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 microM, 0.25+/-0.15 microM 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 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-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-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-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.

Adenylyl Cyclases↗
PubMed1996

Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase.

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the alpha- and beta-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1, 2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 microM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.

Amino Acid Sequence↗
PubMed1995

Co-localization and functional coupling of creatine kinase B and gastric H+/K(+)-ATPase on the apical membrane and the tubulovesicular system of parietal cells.

Immunogold labelling of creatine kinase B (BB-CK) and gastric H+/K(+)-ATPase in the parietal cells of the stomach revealed colocalization of these two enzymes on the apical membrane and the membranes of the tubulovesicular system. Upon fractionation of hog parietal cells, a specific fraction of the BB-CK proteins remained associated with the purified vesicles, in which gastric H+/K(+)-ATPase is highly enriched. The BB-CK present in this highly purified preparation was able to support pronounced H+/K(+)-ATPase activity in K(+)-loaded vesicles in the presence of phosphocreatine and ADP, although only low levels of ATP were measured. In contrast, when pyruvate kinase, phosphoenolpyruvate and ADP were used as an ATP-generating system to sustain similar levels of H+/K(+)-ATPase activity, ATP levels were more than 10-fold higher. Changing the experimental conditions such that ATP levels were the same for both systems resulted in significantly elevated H+/K(+)-ATPase activities in the BB-CK/phosphocreatine system in comparison with the pyruvate kinase/phosphoenolpyruvate system. These results indicate that gastric H+/K(+)-ATPase has preferential access to ATP generated by creatine kinase co-localized on the membranes of the vesicles.

Adenosine Diphosphate↗
PubMed1995

Ethanol stimulates expression of functional H+,K(+)-ATPase in SF9 cells.

The baculovirus expression system is suitable for functional expression of gastric H+,K(+)-ATPase. Expression of functional H+,K(+)-ATPase in Sf9 cells is accompanied by synthesis of large amounts of non-functional subunits. When H+,K(+)-ATPase is synthesised in the presence of 150-250 mM ethanol in the culture medium, two to threefold higher levels of functional H+,K(+)-ATPase are produced due to the formation of more functional subunits rather than to an increase of subunits per se. The catalytical properties of the ethanol-produced H+,K(+)-ATPase are indistinguishable from control preparations. The mechanism by which ethanol stimulates the formation of functional H+,K(+)-ATPase probably involves a direct effect on the physical properties of Sf9 membranes. In addition there also might be an indirect effect through ethanol inducible stress proteins acting as molecular chaperones.

Alcohols↗
PubMed1995

Sodium acts as a potassium analog on gastric H,K-ATPase.

The effects of Na+ on gastric H,K-ATPase were investigated using leaky and ion-tight H,K-ATPase vesicles. Na+ activated the total ATPase activity in the absence of K+, reaching levels of 15% relative to those in the presence of K+. The Na+ activation, which takes place at the luminal side of the membrane, depended on the ATP concentration and the type of buffer used. The steady-state ATP phosphorylation level, studied with leaky vesicles, was reduced by Na+ due to both activation of the dephosphorylation reaction and a shift to E2 in the E1<==>E2 equilibrium. By studying this equilibrium in ion-tight H,K-ATPase vesicles, it was found that Na+ drives the enzyme via a cytosolic site to the nonphosphorylating E2 conformation. No H(+)-like properties of cytosolic Na+ could be detected. We therefore conclude that Na+ behaves like K+ rather than like H+ in the H,K-ATPase reaction.

Adenosine Triphosphate↗
PubMed1994

[Severe hyponatremia, possibly due to inappropriate antidiuretic hormone secretion, during use of the antidepressant fluoxetine].

In a 78-year-old female with hyponatraemia, the syndrome of inappropriate ADH secretion as a side effect of fluoxetine, a relatively new antidepressive drug, was tentatively diagnosed. In the literature 14 cases of this adverse reaction have been described. One should be careful when prescribing fluoxetine to elderly persons, notably when a diuretic is prescribed as well.

Aged↗
PubMed1994

Tertiary amines as antagonists of both the luminal and cytosolic K(+)-site of gastric H,K-ATPase.

Tertiary amines like imidazole and triallylamine lower the apparent affinity of K+ in the ATP hydrolysis reaction of pig gastric H,K-ATPase in a pH and amine concentration dependent way. The mechanism and sidedness of this effect was studied by analyzing the partial reactions of the enzyme in both leaky and ion-tight vesicles. In leaky vesicles Tris and Hepes had nearly no effect on the apparent Km for K+ in the ATPase reaction, but imidazole (Ki = 13 mM) and triallylamine (Ki = 1.6 mM) markedly decreased the K+ affinity. The steady-state ATP-phosphorylation level in the absence of K+ was not or only slightly affected by these compounds. The reduction of the ATP-phosphorylation level by K+, however, again depended on both the type and concentration of tertiary amine used. A comparable K(+)-amine antagonism was observed in the dephosphorylation reaction. In tightly sealed vesicles, where no activation of K+ at the luminal side could occur, K+ reduced the affinity for ATP in the phosphorylation reaction. Triallylamine counteracted this effect. The K(+)-activated p-nitrophenylphosphatase activity in these ion-tight vesicles also showed a K(+)-triallylamine antagonism. Inhibition of H,K-ATPase activity in these vesicles by triallylamine was immediate (with nigericin present in order to allow intravesicular K+ activation), suggesting the transmembrane feature of this inhibition. These results indicate that tertiary amines decrease the affinity for K+ at both luminal and cytosolic binding sites by interaction at the cytosolic side of the membrane. This results in shifts in the equilibrium of both the E1.H<==>E1.K transition and in the dephosphorylation reaction, E2-P-->E2.K.

Adenosine Triphosphate↗
PubMed1994

Double blind field evaluation of a trivalent vaccine against respiratory disease in veal calves.

A field trial was performed to determine the effect of a trivalent vaccine on Clinical Respiratory Tract Problems (CRTP) in veal calves. The vaccine has been developed to increase immunity against the causal agents of IBR, BVD and BRSV infection. In total 928 calves, housed in 16 compartments of one herd, were involved. In four compartments of 58 calves each, vaccinated and non-vaccinated calves were housed together. Four other compartments were treated as a whole and 8 compartments were left untreated. CRTP incidence, medications, weight gain, haemoglobulin and IgG level were recorded. From CRTP positive animals, seroconversion and presence of specific bacteriae and/or viruses were measured as well. Results of the compartments where vaccinated and non-vaccinated calves were housed together showed that the incidence of CRTP in vaccinated calves was 0.16 while it amounted to 0.28 in controls. Most cases were found between day 40 and 60 after the start of the trial. Seroconversion for vaccine specific viruses was sporadically found, but the presence of Pasteurella's was confirmed in the majority of cases. Presumably, the higher incidence of CRTP in the control group was due to a higher level of BVDV infection which might facilitate a clinical infection with Pasteurella's. Vaccination was also negatively related to the percentage of affected lungs at slaughter, the number of days antibiotics had to be administered and subsequently to medicine costs, although these effects were not significant. Daily weight gain was significantly affected by CRTP, but not by vaccination. The effects of vaccination in the compartments where calves were either all vaccinated or not-vaccinated, were similar or larger when compared to the effects in compartments where half of the calves were vaccinated.

Animals↗
PubMed1994

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.

Animals↗
PubMed1993

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 alpha- as well as the beta-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 native 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.

Animals↗
PubMed1992

Evaluation of an atrophic rhinitis vaccine under controlled conditions.

A vaccine containing inactivated cultures of Bordetella bronchiseptica, toxigenic Pasteurella multocida type D and dermonecrotic P multocida type D toxoid in an oil-in-water adjuvant was given to seven sows, with seven others acting as controls. Half the piglets in each litter were exposed intranasally when four days old to B bronchiseptica and when eight days old to toxigenic P multocida type D. There was considerably less sneezing in the litters of the vaccinated sows and when the piglets were 10 weeks old, only 18 per cent had deformed snouts compared with 74 per cent in the litters of the control sows. The average liveweight gain of the piglets born to vaccinated sows was significantly better (P less than 0.05) between two and 10 weeks of age than that of the piglets born to unvaccinated sows, although there were no significant lower respiratory tract lesions in either group. The conchal atrophy scores were significantly lower (P less than 0.001) in the piglets from the vaccinated sows and were negatively correlated (r = -0.37) with increasing liveweight gain. In the liters of the vaccinated sows, P multocida was not isolated from the nasal passages of the in-contact piglets and from only 7 per cent of those deliberately exposed compared with 65 per cent and 79 per cent, respectively, in the litters of the control sows. P multocida was isolated post mortem from the tonsils of 23 per cent of the piglets of vaccinated sows and from 87 per cent of those from unvaccinated sows.

Animals↗