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R E Forster

Publications and source records attributed to R E Forster.

At least 19 recordsLinked to original sources
PubMed1992

Carbonic anhydrase in the membrane of the endoplasmic reticulum of male rat liver.

We have prepared subcellular fractions of male rat liver homogenate by the method of Lewis and Tata [Lewis, J. A. & Tata, J. R. (1973) J. Cell Sci. 23, 447-459], further purifying the membranes of the microsomal fraction by exposure to 0.01% Triton X-100 and centrifugation. We determined the purity of the fractions with marker enzymes and measured carbonic anhydrase (CA; EC 4.2.1.1) activity in intact and solubilized particulates with 18O exchange between CO2/HCO3- and water. We measured the concentration of CA by titration with a sulfonamide inhibitor, ethoxzolamide, obtaining an average value of 3.8 mumol/mg of microsomal membrane protein. The equilibrium constant for binding ethoxzolamide was 0.49 x 10(-9) M. The Km for CO2 was 1.7 mM and the turnover number was 560,000 sec-1, characterizing this as a membrane-bound, high-activity isozyme of type IV. By electron microscopy of tissue sections after staining with a cobalt precipitation technique, CA was seen in small cytoplasmic vesicles in hepatocytes and in microsomal particles and membranes. There was a sulfonamide-resistant (isozyme type III) and a sulfonamide-sensitive (isozyme type II) CA in the cytosol but none in the rapidly sedimenting endoplasmic reticulum. We conclude that there is no CA normally within the matrix of the cell endoplasmic reticulum but that the CA type III found in the microsome may have been captured from the cytosol during resealing. Thus the adult male rat hepatocyte contains CA type IV in the membrane of the endoplasmic reticulum and CA type II and CA type III in the cytoplasm.

Animals↗
PubMed1992

Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle.

Endurance muscle performance is highly dependent on ATP production from mitochondrial oxidative phosphorylation. To study the role of the mitochondrial oxidative enzymes in muscle fatigue, we analyzed the relationship between the concentrations of substrates associated with ATP synthesis and the muscle performance of electrically stimulated rabbit muscle under CO2-induced acidosis. Two different conditions of pacing-induced muscle performance were produced in the gastrocnemius and soleus muscle groups in anesthetized rabbits by stimulating the sciatic nerve submaximally at two frequencies. Phosphorus nuclear magnetic resonance was used to measure ATP, phosphocreatine, and Pi and to provide data for a calculation of intracellular pH and free ADP. To induce acidosis, the animal was ventilated with 20% CO2. The administration of CO2 effectively reduced the intracellular pH from 6.9 to 6.7 and reduced the isometric tension-time integral (TTI) to below half the value measured in normocapnia at the low pacing frequency. A twofold increase in the pacing frequency resulted in a doubling of the TTI in normocapnia and a tripling of TTI in hypercapnia. The increases in TTI corresponded with increases in free ADP and Pi concentrations. Under the various conditions, all free ADP values were near the in vitro Michaelis-Menten constant (Km) of ADP. The Michaelis-Menten relationship of the oxidative phosphorylative enzymes was applied to the change in substrate concentrations with respect to TTI. From this relationship we observed that the in vivo Km of free ADP was 26 microM, which is close to the in nitro Km, and that Km and maximal reaction velocity did not change under hypercapnia and increased pacing frequency.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1990

Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase.

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.

Animals↗
PubMed1989

Carbonic anhydrase in guinea pig skeletal muscle mitochondria.

The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.

Animals↗
PubMed1989

Rate of CO uptake by canine erythrocytes as a function of PO2.

We used a continuous-flow rapid-mixing apparatus with spectroscopic analysis to measure the rate of CO uptake by canine erythrocytes at 37 degrees C at five different PO2 values from 0 to 553 Torr. Fresh blood from five different dogs was used for the experiments. PCO approximated 80 Torr. Corrections for the lower capillary PCO during a measurement of the diffusing capacity of lung CO, as made by Roughton and Forster in 1957 (J. Appl. Physiol. 11: 290-302, 1957), were not used. The regression equation for 1/theta, where theta is milliliters of CO combining for each milliliter of whole blood (capacity 0.2 ml/ml) per minute for a PCO of 1 Torr was 1/theta = 1.45 +/- 0.0042 PO2. This equation is very similar to that for human erythrocytes under the same conditions.

Animals↗
PubMed1989

Carbon monoxide diffusing capacity.

Before measuring single-breath DLCO it is important to: (1) standardize carefully test technique and computation using current ATS guidelines; (2) confirm that computerized systems correctly perform the calculations; and (3) confirm that the reference values selected are consistent with the data produced by the device in the laboratory by comparing measured values with predicted values in several healthy subjects. Interpretation of the test should begin with the following steps: (1) confirmation that the test was properly performed; (2) identification of "noise" factors that may interfere with the interpretation (these may include age, body size, ethnic factors, Hb concentration, COHb, smoking, lung volume, PaO2, exercise, and hormonal effects); and (3) correlation of abnormal findings with clinical data to ensure that they are consistent with the clinical diagnosis.

Carbon Dioxide↗
PubMed1988

Carbonic anhydrase activity of intact carbonic anhydrase II-deficient human erythrocytes.

Intact erythrocytes from subjects with deficiency of blood carbonic anhydrase (CA) II and from normal subjects were assayed for enzyme activity by use of an 18O exchange technique in a solution containing 25 mM (CO2 + NaHCO3) plus 125 mM NaCl. At 25 degrees C and pH 7.4, the catalyzed reaction velocity was 0.32 +/- 0.04 M/s for the CA II-deficient and 1.60 +/- 0.12 M/s for the normal cells, a ratio of 1:5. Under the same conditions at 37 degrees C the relative difference between the CA II-deficient and normal cells was much less: the velocity for the CA II-deficient cells was 0.84 +/- 0.07 M/s and for the normal cells 1.60 +/- 0.32 M/s, a ratio of 1:1.9. Results were comparable for the hemolysates with the NaHCO3 reduced to 85 mM (the corresponding intracellular concentration): at 25 degrees C CA II-deficient cells had a velocity of 0.36 +/- 0.01 M/s compared with 1.12 +/- 0.04 M/s for the normal cells, a ratio of 1:3.1. At 37 degrees C again the relative difference between hemolysates from CA II normal and deficient cells was much less: the CA II-deficient cells had a reaction velocity of 1.17 +/- 0.22 M/s vs. 2.60 +/- 0.36 M/s for the normal cells, a ratio of 1:2.2. The greater fractional reduction of enzyme velocity of CA II-deficient cells at 25 degrees C compared with 37 degrees C appears to be explained by a greater chloride inhibition of the presumed CA I at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

A micromethod for measuring carbonic anhydrase activity using 18O exchange between CO2 and H2O.

We have developed a method of measuring the activity and characteristics of carbonic anhydrase (CA) using the disappearance of 18O from CO2 in 1 ml of gas contained in a glass chamber as it exchanges with H2O in 0.01 ml 0.25 M NaHCO3 solution in a thin (25 micron) porous membrane. Serial gas samples (approximately 0.02 ml) are analyzed in a mass spectrometer to obtain the rate of disappearance of the label. The enzyme activity can be measured inside intact cell or particle membranes. As little as 10(-15) mol of high-activity type CA can be detected at 25 degrees C, and the activity of 200 times this amount can be measured. The uncatalyzed hydration reaction velocity constant was 0.056 +/- 0.004 s-1, in agreement with published data.

Bicarbonates↗
PubMed1978

Bicarbonate-chloride exchange in erythrocyte suspensions. Stopped-flow pH electrode measurements.

A pH-sensitive glass electrode was used in a temperature-controlled stopped-flow rapid reaction apparatus to determine rates of pH equilibration in red cell suspensions. The apparatus requires less than 2 ml of reactants. The electrode is insensitive to pressure and flow variations, and has a response time of < 5 ms. A 20% suspension of washed fresh human erythrocytes in saline at pH 7.7 containing NaHCO(3) and extracellular carbonic anhydrase is mixed with an equal volume of 30 mM phosphate buffer at pH 6.7. Within a few milliseconds after mixing, extracellular HCO(3) (-) reacts with H(+) to form CO(2), which enters the red cells and rehydrates to form HCO(3) (-), producing an electrochemical potential gradient for HCO(3) (-) from inside to outside the cells. HCO(3) (-) then leaves the cells in exchange for Cl(-), and extracellular pH increases as the HCO(3) (-) flowing out of the cells reacts with H(+). Flux of HCO(3) (-) is calculated from the dpH/dt during HCO(3) (-)-Cl(-) exchange, and a velocity constant is computed from the flux and the calculated intracellular and extracellular [HCO(3) (-)]. The activation energy for the exchange process is 18.6 kcal/mol between 5 degrees C and 17 degrees C (transition temperature), and 11.4 kcal/mol from 17 degrees C to 40 degrees C. The activation energies and transition temperature are not significantly altered in the presence of a potent anion exchange inhibitor (SITS), although the fluxes are markedly decreased. These findings suggest that the rate-limiting step in red cell anion exchange changes at 17 degrees C, either because of an alteration in the nature of the transport site or because of a transition in the physical state of membrane lipids affecting protein-lipid interactions.

Bicarbonates↗
PubMed1978

Influence of pH on elastic deformability of the human erythrocyte membrane.

Fresh human blood was diluted 1:5000 in buffered saline-sucrose solution and titrated to a pH varying from 4.5 to 10.5 with 0.1 N HCl or 0.1 N NaOH. Circular regions of the membrane of individual cells were then deformed at 25 degrees C by aspiration into a micropipette having an internal tip diameter of 0.9-1.4 micron. A membrane surface elasticity modulus, mu (dyn/cm), was computed from the relationship between length of the aspirated membrane and the deforming pressure according to a two-dimensional membrane model. Surface elasticity increases with decreasing pH and with time after the cell suspension is acidified, rising several orders of magnitude with a t1/2 of 1--5 h as pH is lowered from 7.2 to 4.6. This increase in mu is only partially reversible. pH greater than 7.2 had little effect on mu. Membrane surface elasticity is not affected by variations in external [Ca2+] over the range of 0--50 mM, tonicity of the suspension medium from 275--400 mosM, or age of 0--50 h. Addition of 50 mM NaHCO3 to the medium increases the rate of change of mu at a given pH. These results suggest that the elastic properties of the red cell membrane are largely determined by interactions among structural proteins located on the cytoplasmic surface of the membrane and that these interactions are initiated by changes in intracellular pH.

Elasticity↗
PubMed1978

Analysis of postcapillary pH changes in blood in vivo after gas exchange.

A quantitative description of the reaction and transport processes that take place in blood during and after gas exchange in capillaries is developed and used to interpret recently reported experimental results. Included in the computation are 1) CO2-H2CO3 hydration-dehydration reactions in plasma and erythrocytes, 2) CO2 reactions with hemoglobin, 3) O2 binding to hemoglobin, 4) buffering of H+ intra- and extracellularly, 5) HCO3- Cl- exchange across the red cell membrane, 6) diffusion of gases between alveolar gas and blood, and 7) transcellular movement of water. Ion and water fluxes are described assuming passive diffusion down their electrochemical potential gradients. Recent data on the magnitude of the Bohr and Haldane shifts and on carbamate formation in the presence of 2,3-diphosphoglycerate are used. The analysis is used to examine the direction, magnitude, and time course of plasma pH changes in blood leaving the pulmonary capillaries and is shown to preduct results that agree very closely with recently reported experimental measurements in vivo. The time computed for plasma pH equilibration after gas exchange when carbonic anhydrase activity is absent from plasma is so great that blood may never be in complete electrochemical equilibrium as it travels around the circulation in normal man.

Bicarbonates↗
PubMed1977

Carbonic anhydrase activity in intact red blood cells measured with 18O exchange.

We have used a stirred, temperature-regulated, reaction vessel separated by a Teflon membrane from the ion source of a mass spectrometer to monitor continuously the time course of disappearance of C18O16O, mass 46, at chemical equilibrium as the 18O exchanges with 16O in water. This instrument is sensitive to less than 0.01 mm Hg of partial pressure of C18O16O with a response time of less than 3 s. The equation of Mills and Urey was used to calculate the hydration velocity constant for uncatalyzed or catalyzed homogenous solutions from the exponential disappearance of mass 46. Addition of red blood cells to the reaction mixture produces biphasic (double exponential) disappearance curve for mass 46. A theory of this process has been developed which describes the time course of [C18O16O] as a function of the catalytic factor for intracellular carbonic anhydrase (A) and the permeability of the cell membrane to HCO3- (P) in addition to the known values; water volume of the cells in the suspension, extracellular pH, the extracellular hydration reaction velocity constant, ku, and dehydration reaction velocity constant, ku. Using this theory, A and P were estimated from the disappearance curve for mass 46 at different values of hematocrit in the reaction mixture, both by a trial and error curve fitting procedure and by a more convenient graphical linearization method. The values of A and P obtained were very sensitive to small amounts of lysis (less than 1%), but the graphical method of analysis minimized this effect. For the blood cells of five normal subjects suspended in 24 mM bicarbonate in 145 mM NaCl at pH 7.4 and 37 degrees, using the graphical method we obtained an average value of 9,906 for A as compared to 19,900 for a comparable concentration of hemolysate. Correcting for a lower pH and chloride concentration inside the cell the latter figure would reduce to 17,500, still 80% higher than the intracellular value. The reason for this discrepancy is not clear. The average permeability of the red cell to bicarbonate ion was 3 X 10(-4) cm/s.

Adult↗
PubMed1977

Can alveolar pCO2 exceed pulmonary end-capillary CO2? No.

The exchanges and chemical reactions of CO2 within the blood are not complete during its transit time through the alveolar capillaries, so that theoretically alveolar PCO2 cannot exactly equal end-capillary blood PCO2. However, as a practical matter, the technical errors in determining alveolar and peripheral arterial PCO2 are so large in comparison with the difference between pulmonary arterial PCO2 and pulmonary venous PCO2, owing to the large effective solubility of CO2 in blood, that for this reason alone any difference between alveolar and end-capillary PCO2 can be neglected. The hypothetical mechanism that was originally proposed by Gurtner, Song, and Farhi for the production of an alveolar PCO2 greater than end-capillary PCO2 during rebreathing and depends on the existence of a radial gradient of H+ X HCO3- in the capillary blood near the wall appears unlikely because a) it would require a significant separation of charged ions over a large proportion of the capillary length and for a large proportion of the blood transit time and b) it would require a large radial gradient of PCO2 within the blood which on thermodynamic grounds would in turn require an unreasonable amount of energy to maintain.

Animals↗
PubMed1977

Postcapillary changes in blood pH in vivo during carbonic anhydrase inhibition.

A rapidly responding stopped-flow glass pH electrode apparatus was used to investigate pH changes in blood in vivo after it exits from an exchange capillary. Arterial blood was drawn from anesthetized animals through the apparatus. Temperature and pH of the blood in the electrode chamber were continuously recorded, both during withdrawal and after flow was stopped. Blood pH did not change after stopping flow in control experiments. When benzolamide (2 mg/kg) was given to inhibit carbonic anhydrase activity available to plasma (e.g., due to lysis) while having less effect on intracellular activity, pH increased 0.02-0.04 (t1/2 approximately 8 s) after stopping flow. Administration of acetazolamide (50 mg/kg) resulted in pH decreasing 0.07-0.10 (t1/2 approximately 15 s) after stopping flow. Ventilation for 1 min with N2 resulted in an increased rise in pH for the benzolamide-treated animals but a decreased fall in pH for the acetazolamide-treated animals. These shifts in arterial blood pH after gas exchange are largely due to disequilibrium of [H+] between red cells and plasma at the end of the pulmonary capillary.

Acetazolamide↗