Biomedical subjects

J Voigt

Publications and source records attributed to J Voigt.

At least 37 records · Page 2Linked to original sources

PubMed1999

Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor.

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.

Cell Line↗

PubMed1999

Quenching of chlorophyll fluorescence by triplets in solubilized light-harvesting complex II (LHCII).

The quenching of chlorophyll fluorescence by triplets in solubilized trimeric light harvesting complexes was analyzed by comparative pump-probe experiments that monitor with weak 2-ns probe pulses the fluorescence yield and changes of optical density, DeltaOD, induced by 2-ns pump pulses. By using a special array for the measurement of the probe fluorescence (Schödel R., F. Hillman, T. Schrötter, K.-D. Irrgang, J. Voight, and G. Biophys. J. 71:3370-3380) the emission caused by the pump pulses could be drastically reduced so that even at highest pump pulse intensities, IP, no significant interference with the signal due to the probe pulse was observed. The data obtained reveal: a) at a fixed time delay of 50 ns between pump and probe pulse the fluorescence yield of the latter drastically decreased with increasing IP, b) the recovery of the fluorescence yield in the microseconds time domain exhibits kinetics which are dependent on IP, c) DeltaOD at 507 nm induced by the pump pulse and monitored by the probe pulse with a delay of 50 ns (reflecting carotenoid triplets) increases with IP without reaching a saturation level at highest IP values, d) an analogous feature is observed for the bleaching at 675 nm but it becomes significant only at very high IP values, e) the relaxation of DeltaOD at 507 nm occurs via a monophasic kinetics at all IP values whereas DeltaOD at 675 nm measured under the same conditions is characterized by a biphasic kinetics with tau values of about 1 microseconds and 7-9 microseconds. The latter corresponds with the monoexponential decay kinetics of DeltaOD at 507 nm. Based on a Stern-Volmer plot, the time-dependent fluorescence quenching is compared with the relaxation kinetics of triplets. It is shown that the fluorescence data can be consistently described by a quenching due to triplets.

Biophysical Phenomena↗

PubMed1999

Passage of ribonucleic acid along the intestine of sheep.

Sheep (Flemish female x Texel male, 55 kg BW), fitted with a PVC cannula in the dorsal rumen and single T-shaped PVC cannulas in the proximal duodenum, distal duodenum, mid-jejunum and terminal ileum were fed hay or hay-concentrate diets at various levels of nitrogen and cell walls (NDF) (22 to 32 g N/d; 150 to 699 g NDF/d). Co-EDTA and Cr-NDF were used as markers to measure the flow rate of digesta. Ribonucleic acid (RNA) intestinal digesta and in rumen bacteria was determined with orcinol after extraction with sodium chloride, precipitation with tungstophosphoric acid and alkaline hydrolysis. The RNA:total N ratio in bacteria, harvested from the rumen, amounted to 0.70 (CV 4.4%). The apparent digestibility of RNA in different sections of the intestine was higher than of total N. About 6% of RNA entering the duodenum disappeared between the proximal and distal duodenum. At jejunum, the net disappearance of RNA amounted to 68% of the quantity which entered the proximal duodenum. A higher result of 71% was obtained at the ileum. Total net disappearance of RNA between the proximal duodenum and rectum averaged 75%. Sixteen percent of RNA leaving the ileum was apparently digested in the large intestine. The true digestibility of RNA between the proximal duodenum and the terminal ileum, as estimated by multiple regression analysis, amounted to 78%. Of the amount of RNA entering the ileum, 24% was of endogenous origin. At ileum, the RNA passage was positively related to the ileal flow of NDF (R2 = 0.67) and N (R2 = 0.94). The passage of RNA increased by 3 mg RNA per g ileal indigestible NDF. Ileal endogenous N consisting of approximately 2% of endogenous RNA-N. In conclusion, the digestion capacity in the first part of the small intestine is high. Rising flows of indigestible cell walls and nitrogen increase the loss of ileal RNA. Further, using RNA as a microbial marker to assess the amount of microbial protein entering the duodenum of ruminants, digesta samples should be collected immediately post pylorus at the proximal duodenum, in order to avoid underestimation of the microbial protein synthesis in the rumen.

Animals↗

PubMed1999

Morphological and functional development of the rumen in the calf: influence of the time of weaning. 1. Morphological development of rumen mucosa.

The objective of this study was to determine whether the nutritional regimen of rearing calves would influence the morphometric and histological development of rumen mucosa. Twelve male Holstein calves 7 d of age were assigned to three groups of 4 animals each: milk group (I), early weaned (6 weeks) group (II) and late weaned (9 weeks) group (III). All animals received additional solid feed. Animals of group I were slaughtered after 6 weeks of age, whereas those in groups II and III were slaughtered after 9 weeks of age. At slaughter, the ruminal digesta amounted to 2035 g (milk group), 3092 g (late weaned group) and 5374 g (early weaned group). The differences in the ruminal molar percentage of SCFA were not significant. There was a trend for lower pH and higher SCFA concentrations in the order late weaned, early weaned and milk fed animals (pH: 6.4, 6.6 and 6.7, respectively; SCFA: 96, 87 and 77 mmol/l, respectively). The mean length (1.07 mm in milk group, 1.45 mm in late weaned group and 1.87 mm in early weaned group), width (0.43, 0.58 and 0.71 mm, respectively) and surface of papillae (190, 232 and 241 mm2/cm2 mucosa, respectively) increased with both the age of the animals and the elevated intake of solid feed, whereas the number of papillae (210, 140 and 92 per cm2 mucosa, respectively) decreased. In both milk-fed groups type A and B corneal cells were present in the Stratum corneum, whereas in the earlier weaned calves type C-cells could be also seen. These findings indicate a more advanced stage of development of the rumen epithelium in the earlier weaned calves fed higher amounts of concentrate and hay.

Animal Feed↗

PubMed1999

Calculation of utilizable crude protein at the duodenum of cattle by two different approaches.

The duodenal flow of utilizable crude protein (crude protein minus endogenous protein) in cows was estimated using dietary parameters, first by multiple regression and secondly by the addition of microbial protein and undegraded feed protein. These estimates were compared with 327 results from experiments conducted with fistulated cows in Braunschweig-Völkenrode and Rostock-Dummerstorf. The regressions and the measurements for microbial protein synthesis as well as feed protein degradation and organic matter fermentation in the rumen were based on the same experimental data set. The prediction of utilizable crude protein (uCP) at the duodenum by regression with digested organic matter (kg DOM) and undegraded feed protein (g UDP) as predicting variables, was more accurate than the value given by microbial protein synthesis and rumen protein degradability. The regression model [g uCP = [188.5-(116.5 (UDP/CP))] DOM + 1.03 UDP] had the highest coefficient of determination (r2 = 0.91) and the lowest coefficient of variation (cv = 8.6); indicating the model's superiority over the other method of estimation.

Animals↗

PubMed1998

Rate of carotenoid triplet formation in solubilized light-harvesting complex II (LHCII) from spinach.

In the present study the rate of triplet transfer from chlorophyll to carotenoids in solubilized LHCII was investigated by flash spectroscopy using laser pulses of approximately 2 ns for both pump and probe. Special attention has been paid to calibration of the experimental setup and to avoid saturation effects. Carotenoid triplets were identified by the pronounced positive peak at approximately 507 nm in the triplet-singlet difference spectra. DeltaOD (507 nm) exhibits a monoexponential relaxation kinetics with characteristic lifetimes of 2-9 micros (depending on the oxygen content) that was found to be independent of the pump pulse intensity. The rise of DeltaOD (507 nm) was resolved via a pump probe technique where an optical delay of up to 20 ns was used. A thorough analysis of these experimental data leads to the conclusion that the kinetics of carotenoid triplet formation in solubilized LHCII is almost entirely limited by the lifetime of the excited singlet state of chlorophyll but neither by the pulse width nor by the rate constant of triplet-triplet transfer. Within the experimental error the rate constant of triplet-triplet transfer from chlorophyll to carotenoids was estimated to be kTT > (0.5 ns)-1. This value exceeds all data reported so far by at least one order of magnitude. The implications of this finding are briefly discussed.

Biophysical Phenomena↗

PubMed1998

Influence of dietary concentrate to forage ratio on the development of rumen mucosa in calves.

Effects of structural and non-structural carbohydrates on the development of rumen fermentation and ruminal mucosa in calves were examined during the weaning period. Barley/soybean meal (SBM) group was fed a concentrate starting from 2 weeks of age, whereas alfalfa group received a mixture of concentrate and alfalfa hay in which the proportion of the latter was gradually increased from 20% to 70% between weeks 2 and 9 of age. The total volatile fatty acid concentration in rumen fluid of calves increased with age, but at 9 weeks there were no significant differences between the two diets (barley/SBM group 153 mmol/l, alfalfa group 150 mmol/l). Rumen papillae at 9 weeks of age, as compared to 6 weeks of age, were longer and fewer in number per square centimetre mucosa, with larger cut surface. This resulted in a higher surface of papillae per square centimetre mucosa at 9 weeks (barley/SBM group 286 mm2/cm2, alfalfa group 245 mm2/cm2) than at 6 weeks of age (barley/SBM group 217 mm2/cm2, alfalfa group 198 mm2/cm2). At 9 weeks of age, the pH (barley/SBM 5.0, alfalfa 5.7), the acetate to propionate ratio (barley/SBM 2.2, alfalfa 3.2) as well as the length of the papillae in the ventral ruminal sac (barley/SBM 1.96 mm, alfalfa 2.37 mm) were increased in the alfalfa group when compared to the barley/SBM group (P < 0.1). In the former group, the proportion of butyrate revealed significantly increased values at 4 and 6 weeks of age. In animals of the barley/SBM group at 9 weeks of age, characteristic protrusions with proliferated thick epithelium occurred on the papillae and increased the surface for absorption. On the epithelium (Stratum corneum) desquamating cells with parakeratosis could be observed. In the alfalfa group the papillae of the ventral ruminal sac were longer, without protrusions. The morphotypes of the adhering rumen microflora differed between the groups. It can be concluded that feeding greater amounts of non-structural carbohydrates increases the surface for absorption of the rumen epithelium in calves. The absence of hyperkeratosis and rumenitis in the barley/SBM group indicated that there is no reason to limit high starch diets in the early weaning period of calves.

Acetic Acid↗

PubMed1997

Calorimetric validation of 13C bicarbonate and doubly labeled water method for determining the energy expenditure in goats.

The purpose of the present study was to validate the 13C bicarbonate method (13C-M) and the doubly labeled water method (DLWM) for the estimation of the CO2 production R(CO2) in goats as a ruminant model. Indirect calorimetry was chosen as the reference method. Studies were carried out in 2 male African dwarf goats at 3 different developing stages (age: 5, 10, and 14 months, body mass: 14.6, 20.3, and 21.7 kg). Animals were fed a balanced feed 14 days before and during the studies. The isotope tracers (4 mg/kg NaH13CO3, 120 mg/kg 2H2O, and 75 mg/kg H218O; 99 AT.-%) were simultaneously given as a single pulse injection into the jugular vein. Thereafter, the animals were kept for 8 days in two respiration chambers (volume of chamber: 2.85 m3, air flow rate: 25 1/min) for the estimation of CO2 production and O2 consumption. For the determination of R(CO2) using the 13C-M samples of exhaled breath were drawn from the respiration chambers. The 13C enrichment and CO2 concentration of breath samples were measured by means of an infrared isotope analyzer. In order to determine R(CO2) by means of the DLWM, blood serum was used. The 2H and 18O enrichments were measured by an isotope ratio mass spectrometer. Urine samples were collected over 24 h to quantify renal water losses. The R(CO2) was calculated by means of the 13C-M using the area under the 13C enrichment-time curve. The determination of R(CO2) by means of the DLWM was based on the slopes of the 2H and 18O disappearance curves and the body water pool obtained from the zero time intercept of the isotope curves. The values of R(CO2) resulting from the 13C-M were found to be comparable with those from the calorimetric measurement. Smaller (not statistically significant) values of R(CO2)--92% from 13C-M and 87% from DLWM--compared to the indirect calorimetry could indicate the incorporation of 13C and 2H into metabolites other than CO2 and H2O, respectively. The body water contents calculated from the zero time intercepts of the 2H and 18O disappearance curves amounted to 66% and 63%, respectively. The body water content was found to be not related to the age of animals. The renal water loss was calculated to be 35% of the total water loss (0.76 l/d.

Aging↗

PubMed1997

Production of cell wall polypeptides by different cell wall mutants of the unicellular green alga Chlamydomonas reinhardtii.

Three classes of cell wall-defective mutants of the unicellular green alga Chlamydomonas reinhardtii have been described in the literature differing with respect to the amounts of cell wall material and its attachment to the plasma membrane, respectively. We have compared the production of the chaotrope-soluble cell wall polypeptides by the different mutants. These experiments have been performed by comparative Western-blot analyses using antibodies raised (1) against the deglycosylation products of the insoluble wall fraction of wild-type cells, (2) against the deglycosylation product of the '150 kDa' chaotrope-soluble cell wall polypeptide and (3) against the carbohydrate side chains of the Chlamydomonas cell wall glycoproteins, respectively. Considerably different levels of cell wall polypeptides were found in the LiCl-extracts from intact cells of the various mutant strains containing the apoplastic, chaotrope-soluble cell wall glycoproteins. No correlation was found between the amounts and the patterns of cell wall glycoproteins present in the LiCl-extracts and the electron microscopical classification of the mutant strains. All the mutant strains were shown to contain the same amounts and patterns of intracellular cell wall precursors as wild-type cells as revealed by Western-blot analyses of urea-SDS lysates of LiCl-pretreated cells. These findings indicate that the different mutant strains produce the same set of cell wall polypeptides at the same relative amounts as wild-type cells. However, in the case of some strains belonging to different classes of cell wall mutants and showing differential seggregation patterns in crosses, alterations were observed for the pattern of extracellular cell wall polypeptides present in the LiCl-extracts from intact cells and in the culture medium, respectively.

Animals↗

PubMed1997

Secretion and reabsorption of endogenous protein along the small intestine of sheep: estimates derived from 15N dilution of plasma non-protein-N.

Sheep fitted with a PVC cannula in the dorsal rumen, a silastic infusion tube in the abomasum, and single "T'-shape PVC cannulas in the proximal duodenum (D1), distal duodenum (D2), mid-jejunum (J), and terminal ileum (I), were fed on diets varying in crude protein (N x 6.25) and fibre contents. 15N was administered to the sheep as a 15N-labelled grass meal-beer yeast suspension which was continuously infused into the abomasum over a period of 1 week. Subsequently, over a period of 4 d, 15N excess in the D1, D2, J and I digesta and faeces was measured and related to 15N excess in blood plasma TCA-soluble N. Average daily intakes of DM and N were 1.12 kg and 26.3 g respectively. Average daily endogenous N (g) amounted to 3.1 at D1, 10.2 at D2, 6.1 at J, 5.0 at I, and 5.1 in the faeces (F). Corresponding percentage values for total N were: D1 12.4, D2 32.9, J 46.4, I 48.4 and F 50.7. The secretion, particularly of pancreatic juice and/or bile, varied. The apparent re-absorption of endogenous protein varied among experimental diets, on average by 50.4% between D2 and I. Along the small intestine the percentage of 15N in the TCA-precipitable fraction gradually changed, on average by 66.0, 63.0, 46.3 and 55.7 at D1, D2, J and I respectively. Because of differential rates of enrichment and turnover among protein pools which contribute towards endogenous secretion, care should be taken in interpreting these data as absolute values.

Animal Feed↗

PubMed1996

Kinetics of excited states of pigment clusters in solubilized light-harvesting complex II: photon density-dependent fluorescence yield and transmittance.

Relative fluorescence yield, phi F, and transmittance, T, were measured in solubilized light-harvesting complex II (LHCII) as a function of photon density, Ip, of monochromatic 645-nm laser pulses (duration: approximately 2.5 ns). Special efforts were made in constructing an optical set-up that allows the accurate determination of the fluorescence from an area of constant Ip, phi F(Ip) starts to decline at approximately 10(14) and drops to values below 0.01% at maximum Ip (approximately 10(19) photons cm-2 pulse-1). T(Ip) decreases only slightly at photon densities of approximately 10(15) but increases steeply at values of > 10(17) photons cm-2 pulse-1. The interpretation of the phi F(Ip) data using the saturation limit of Mauzerall's multiple hit model leads to a unit size of about 10-15 chlorophyll molecules. One interpretation is to attribute this result to a very fast exciton-exciton annihilation of multiple excited states generated within this small domain. Alternatively, based on the assumption that delocalized cluster states within the monomeric/trimeric subunit of LHCII exist, the results can be consistently described by a kinetic model comprising ground, monoexcitonic, and biexcitonic states of clusters and a triplet state that is quenched by carotenoids in LHCII. Within the framework of this model the annihilation of multiple excitations is explained as ultrafast radiationless relaxation of higher excited cluster states. Comparative measurements in diluted acetonic Chl a solution are consistently described by the depletion of the ground state, taking the absorption cross section at the used wavelength.

Chlorophyll↗

PubMed1996

Immunological identification of a putative precursor of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

To identify precursors of the insoluble glycoprotein framework of the Chlamydomonas cell wall, a polyclonal antibody was raised against the mixture of polypeptides released from the insoluble wall fraction by chemical deglycosylation. This antibody preferentially cross-reacted with a '150 kDa' salt-soluble cell wall glycoprotein. The conclusion that this '150 kDa' glycoprotein is a putative precursor of the insoluble cell wall fraction was corroborated by the results of pulse-chase experiments and by experiments with antibodies raised against the '150 kDa' salt-soluble glycoprotein and against its 100 kDa deglycosylation product, respectively. Whereas the antibody against the '150 kDa' glycoprotein preferentially recognized carbohydrate side chains, the antibody against its 100 kDa deglycosylation product was found to have essentially the same specificity towards glycosylated and deglycosylated cell wall components as the antibody against the deglycosylation products of the insoluble wall fraction. Furthermore, the antibody against the deglycosylated, insoluble wall fraction recognized almost the same set of peptide fragments derived by V8 protease treatment from the '150 kDa' salt-soluble cell wall glycoprotein and its 100 kDa deglycosylation product, respectively, as the antibody against the 100 kDa deglycosylated cell wall polypeptide.

Amino Acid Sequence↗

PubMed1995

A Chlamydomonas homologue to the 14-3-3 proteins: cDNA and deduced amino acid sequence.

We have isolated and sequenced a 1464 bp cDNA from the unicellular green alga Chlamydomonas reinhardtii encoding an acidic polypeptide (259 aa) with considerable homologies to the 14-3-3 proteins of animals, yeasts and higher plants. Like the other members of this highly conserved protein kinase regulatory protein family, the deduced amino acid sequence of the Chlamydomonas 14-3-3 protein includes two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca(2+)-binding sites is located within the C-terminal part of this polypeptide (positions 208-219). EF hand motifs are also present in the 14-3-3 proteins of some higher plants but not in those of animals and yeasts.

14-3-3 Proteins↗

PubMed1995

[Evaluation of different markers for the determination of microbial nitrogen flow into the duodenum of dairy cows].

2,6-Diaminopimelic acid (DAPA), ribonucleic acid (RNA), 15N, D-alanine (D-ALA) and the amino acid profiles (AAP) were compared as microbial markers for determination of the microbial protein synthesis in the rumen. Three dairy cows (Schwarzbuntes Milchrind, LW 602 kg), each fitted with a rumen cannula and a re-entrant cannula in the proximal duodenum, were offered four isoenergetic and isonitrogenous diets (mean daily intake 15.0 +/- 0.45 kg DM; forage: concentrate = 50:50) in a periodic experiment. The diets contained soyabean extracted meal, meat and bone meal, pea meal and dried clover as major sources of protein. On the 4th day after administration of 9 g 15N-labelled urea (95 atom-% 15N-excess) per day, samples of rumen fluid and duodenal digesta were obtained 3 h after feeding. The bacteria were isolated by differential centrifugation. Bacteria harvested from the rumen had significantly higher 15N enrichment and D-ALA: N ratio than 'duodenal' bacteria. However, DAPA: N ratio was higher in 'duodenal' bacteria compared to rumen bacteria. There were no differences in RNA: N ratio between rumen and 'duodenal' bacteria. The source of the bacteria in the digestive tract has an influence on the ratio of microbial N: total N, especially when 15N, AAP, DAPA and D-ALA but not RNA were used as markers. The most reproducible method was D-ALA (C.V. 4.7 for rumen and 6.8 for 'duodenal' bacteria) followed by 15N (10.8 resp. 4.8) and RNA (9.7 resp. 8.2). The results obtained with 15N and D-ALA agreed closely at the same source of bacteria. The RNA method reached the level of these markers (15N, D-ALA) when the bacteria were isolated from the duodenum. It is concluded that D-ALA (bacteria isolated from rumen and duodenum) and also 15N (bacteria isolated from duodenum) were the best markers for estimation of the microbial protein synthesis.

Alanine↗

PubMed1994

[Biliary pseudocyst as a rare complication of choledocholithiasis].

Biliary pseudocysts, so-called biliomas are reported to be an extreme rarity and mostly arising posttraumatic. We report on a case of symptomatic choledocholithiasis 7 years after cholecystectomy. The course was complicated by formation of a biliary pseudocyst. The 71-year-old, septic patient could not be operated because of pulmonal and cardiac high risks. He could be treated successfully by conservative procedure via endoscopic papillotomy, stone extraction and percutaneous, sonographic controlled drainage of the biliary pseudocyst.

Aged↗

PubMed1993

Regulation of the uncoupled GTPase activity of elongation factor G (EF-G) by the conformations of the ribosomal subunits.

The elongation factor G (EF-G) GTPase activity is induced by either 70S ribosomes or 50S ribosomal subunits. The GTPase activity induced by 50S ribosomal subunits is predominant at low concentrations of monovalent cations and decreases with increasing concentrations of K+ or NH4+. Double-logarithmic plots of the data reveal straight lines with different slopes for low and high concentrations of monovalent cations, respectively, intersecting at the same concentration of monovalent cations where maximal EF-G GTPase activity is measured in the presence of both ribosomal subunits. Substantially the same curves are obtained when 50S ribosomal subunits are substituted by 50S CsCl-core particles partially reconstituted by addition of purified 50S split proteins L7/L12. Intact 30S ribosomal subunits, but not 30S CsCl-core particles are able to associate with 50S ribosomal subunits and to modulate ribosome-dependent EF-G GTPase activity. Therefore, our data clearly show that the biphasic courses of the NH4+ and K+ curves of EF-G GTPase activity induced by 50S ribosomal subunits are not due to contaminations with 30S ribosomal subunits but result from different conformations of EF-G/50S ribosomal-subunit complexes at low and high concentrations of monovalent cations, respectively. CD spectra of 50S ribosomal subunits measured under different salt conditions have shown that the conformation of the 50S ribosomal subunits is strongly dependent on the concentration of monovalent cations. The conformation of 30S ribosomal subunits is, however, considerably stronger influenced by the Mg2+ than by the concentration of monovalent cations. The salt effects on the conformation of the 30S ribosomal subunits correspond to the salt effects on the association of ribosomal subunits and the modulation of EF-G GTPase activity by 30S ribosomal subunits. Since, in the presence of both ribosomal subunits, EF-G GTPase activity is maximal at the same concentration of monovalent cations where obviously a spontaneous conformation change of 50S ribosomal subunits takes place, we postulate that EF-G GTPase primarily acts on the ribosomes by changing the conformation of 50S ribosomal subunits. The resulting model is based on the assumption that EF-G GTPase activity is considerably more strongly induced by the 'substrate conformation' ('state I') than by the 'product conformation' of the 50S ribosomal subunits ('state II'). A spontaneous transformation of 'state II' to 'state I' is expected to occur in the absence of mRNA, aminoacyl-tRNA and EF-T especially under salt conditions favouring state I.

Cations↗

PubMed1993

Regulation of elongation factor G GTPase activity by the ribosomal state. The effects of initiation factors and differentially bound tRNA, aminoacyl-tRNA, and peptidyl-tRNA.

The elongation factor G (EF-G) is responsible for the translocation of the ribosome along the mRNA chain. Under in vitro conditions, EF-G exhibits a very active uncoupled GTPase activity which is dependent on the presence of ribosomes and is modulated by mRNA-dependent binding of tRNA. In the absence of tRNA, uncoupled EF-G GTPase is inhibited by initiation factors IF1 and IF3, but not by initiation factor IF2. In the presence of N-fMet-tRNAfMet and poly(A,U,G) or in the presence of N-acetyl-Phe-tRNAPhe and poly(U), initiation factor IF2 causes an additional decrease of the uncoupled EF-G GTPase activity. This effect, however, is dependent on the presence of IF1 and IF3 and is obviously due to the mRNA- and initiation factor-dependent binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, respectively, to the ribosomal P-site. Non-enzymatic binding of N-fMet-tRNAfMet and N-acetyl-Phe-tRNAPhe, however, causes a stimulation of uncoupled EF-G GTPase activity. The same effects are observed for Met-tRNA, Phe-tRNAPhe and uncharged tRNA. These findings are discussed in the light of the three-site model of the ribosome and the mechanism of translocation.

Chromatography, DEAE-Cellulose↗

PubMed1993

Localization of the glucocorticoid receptor in rat liver cells: evidence for plasma membrane bound receptor.

1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction. 2. After in vivo administration of [3H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes. 3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol). 4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes. 5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.

Animals↗