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Biomedical subjects

W Junge

Publications and source records attributed to W Junge.

At least 91 records · Page 5Linked to original sources
PubMed1988

Renal handling of 125I-labelled homologous pancreatic lipase and amylase in the rat.

Experiments were carried out in vivo on rats and in vitro on tubular brush border vesicles in order to study the renal mechanisms of the elimination of pancreatic lipase and amylase from the circulation. Highly purified 125I-labelled homologous lipase, amylase or 125I-labelled di-iodo-tyrosine was injected intravenously in a single dose. The sieving coefficients of lipase and amylase were found to be 0.126 and 0.118 respectively. Less than 1% of the lipase activity but more than 10% of the radioactivity were found in the urine in the course of a 120 min experiment. In experiments with amylase, 16% of the enzyme activity and 19% of the radioactivity were present in the urine. Elimination of both enzymes showed first order kinetics and was of the same magnitude (17-24 min). The elimination curves of the radioactivity consisted of at least two components: a fast component immediately after the injection, which was identical with the decrease of the resp. enzyme activity; and a slow component (half-life 106 min), which in both cases proved to be identical with the half-life of di-iodo-tyrosine. In experiments with amylase, the excretion of protein-free 125I-activity started later than with lipase. The radioactivity of 125I-labelled lipase was taken up faster by brush-border-vesicles than that of 125I-amylase. Liberation of protein-free 125I-activity from both enzymes occurred at the same rate. At the end of the experiments the kidneys had no lipase or amylase activity, but they contained 5.4% (lipase), 3.8% (amylase) of the injected radioactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1988

Multicenter evaluation of a specific pancreatic isoamylase assay based on a double monoclonal-antibody technique.

Eleven evaluators from nine laboratories in five countries evaluated a new immunoinhibition method for pancreatic isoamylase determination that is as simple to perform as that for total amylase. The precision at low and intermediate activity concentrations was superior, and at high concentrations it equalled that of the wheat-germ inhibitor method. The test was linear to approximately 2000 U/L, depending on the instrumentation used. The percentage salivary isoamylase activities remaining in specimens after reaction with two monoclonal antibodies ranged from 2 to 4.4%. Comparative studies showed good correlation with the wheat-germ inhibitor (r greater than 0.978) and electrophoresis methods (r = 0.920). Hemolysis, lipemia, and bilirubinemia have no effect on results. Interlaboratory studies demonstrated excellent transferability of the method, if instruments are calibrated with the same calibrator. Reference intervals for pancreatic isoamylase are 13 to 64 U/L (25 degrees C), 13 to 83 U/L (30 degrees C), and 17 to 115 U/L (37 degrees C). A clinical evaluation of patients with acute pancreatitis showed that pancreatic isoamylase has a greater clinical sensitivity than total amylase.

Acute Disease↗
PubMed1987

The role of fixed and mobile buffers in the kinetics of proton movement.

We derive a simple expression for the effective diffusion coefficient of protons in Fick's second law, Deff, when both spatially fixed, HF, and mobile, HM, buffers are present. These buffers are present at moderately high concentrations ([Ftot], [Mtot] greater than 1 mM) in most biological systems. We consider only the case where the protonation reactions remain at equilibrium during the diffusion process. When the pH is to the alkaline side of the pK values of the fixed and mobile buffers ([H+] less than KF, KM), the effective diffusion coefficient of protons in Ficks second law is: (Formula: see text) where DH is the diffusion coefficient of the protons free in the aqueous phase and DHM is the diffusion coefficient of the mobile buffer. The equation illustrates three features of diffusion in a buffered system. Firstly, the effective diffusion coefficient of protons is always lower than the diffusion coefficient of free protons. Secondly, increasing the concentration of fixed buffers always decreases Deff. Thirdly, increasing the concentration of mobile buffer can increase Deff when fixed buffers are present.

Biological Transport↗
PubMed1987

Complete tracking of transient proton flow through active chloroplast ATP synthase.

Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CF0-CF1 (where CF0 is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of P(i) and ADP, the electric potential transients elicited transients of proton flow via CF0-CF1, typically 1.3 H(+) per CF1 and flash group. Proton flow was blocked by CF0-CF1 inhibitors: N,N'-dicyclohexylcarbodiimide, acting on the channel component CF0, and tentoxin, acting on the catalytic component CF1. The half-rise time was 40 ms in (1)H(2)O and 78 ms in (2)H(2)O. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a K(m)(P(i)) of only 14 muM, contrasting with a K(m) of several hundred micromolar for continuous ATP synthesis at high rate. This might reflect a resistance to P(i) diffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CF0-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CF0-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.

Journal Article↗
PubMed1986

The proton channel, CF0, in thylakoid membranes. Only a low proportion of CF1-lacking CF0 is active with a high unit conductance (169 fS).

We investigated the conductance of pea thylakoid membranes and their capacity for photophosphorylation as function of the extraction of chloroplast coupling factor CF1. The degree of extraction was varied via the incubation time in EDTA-containing hypo-osmolar medium and was measured by rocket electroimmunodiffusion. The conductance of thylakoid membranes was measured by flash kinetic spectrophotometry. The time course of extraction followed the time course of thylakoid swelling. Contrary to expectation increasing loss of CF1 did not primarily increase the velocity of proton efflux from each vesicle. Instead proton-tight vesicles were converted to leaky ones, which lost phosphorylating activity. Two subpopulations occurred, although both types of vesicles, leaky and proton-tight ones, were CF1-depleted to a similar degree. This implied that only a small fraction of CF1-lacking CF0 was functional as a proton channel. Tight vesicles had no functional channels while leaky ones had at least one. We determined the proportion of tight vesicles in three independent ways: via the residual phosphorylation activity, via measurements of proton efflux and via measurements of the electric relaxation across the membrane. The results obtained were identical. A statistical evaluation of the data led us to the following conclusions. EDTA treatment produced vesicles containing approximately 10(5) chlorophyll molecules, equivalent to a total of approximately 100 CF0CF1 per vesicle. Even at the highest degree of extraction (75% of total CF1 extracted) only 2.5 out of 75 exposed CF0 per vesicle were proton-conducting. The unit conductance of one open CF0 channel was 169 +/- 18 fS at pH 7.5 and room temperature. At an electrical driving force of 100 mV this was equivalent to the passage of approximately 10(5) protons/s. The most important consequence of this relatively high unit conductance was that a single open CF0 channel was capable of dissipating the protonmotive force of one vesicle, thereby deactivating the whole remaining catalytic capacity of this vesicle.

Edetic Acid↗
PubMed1986

Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase. The role of the delta subunit for proton conduction through CF0.

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the delta & epsilon subunits: CF1(-delta) and CF1(-epsilon). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the beta subunit and in separate peaks CF1(-delta) and CF1(-epsilon). Elution from Mono Q in the presence of tenside yielded a beta delta fragment, CF1(-delta) and CF1(-delta epsilon). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-delta) and CF1(-delta epsilon) were active with cross-reactivity between spinach, pea and maize. delta-containing CF1 always reconstituted higher activities than delta-deficient CF1. The beta delta fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while beta and DCCD-inhibited CF1(-delta) were not. These results support the notion that subunit delta can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082].

Chloroplasts↗
PubMed1986

Assessment of titrimetric and photometric methods for the determination of chymotrypsin catalytic activity in stool.

In this report a method for the spectrophotometric measurement of stool chymotrypsin (CT) is presented and compared to the more commonly used titrimetric method. Homogenisation of specimen and dissolution of CT is examined in detail. The advantages of the photometric method lie in the small size of sample required, and its easy adaptation to manual or automated analysis with a CT-specific amide substrate.

Catalysis↗
PubMed1985

The role of the kidney in the elimination of pancreatic lipase and amylase from blood.

Two clinical observations indicate that the kidney plays the main role in the elimination of lipase and amylase from the circulation: 1. in patients with uncomplicated acute pancreatitis the decrease of the activity of both enzymes in the serum ran almost in parallel. The half life for lipase was found to be 6.9-13.7 h, and somewhat higher figures (9.3-17.7 h) were calculated for amylase; 2. in patients with reduced glomerular filtration rate the serum activity of either or of both enzymes was distinctly elevated. The contribution of the kidney to the elimination of lipase and amylase from blood was studied in the rat. After an intravenous bolus injection of homologous lipase and amylase, the serum activity of both enzymes decreased rapidly. The half-life of lipase was 18.1 min, that of amylase 20.5 min. Up to 30% of the injected amylase but only traces of lipase activity were recovered in the urine. In animals with ligated kidneys the serum half-life of both enzymes was 3 times longer. Our results indicate that lipase as well as amylase are removed from the serum mainly by glomerular filtration at nearly the same rate. Reabsorption of lipase is almost complete, in contrast to that of amylase. It is suggested that the differences in the renal handling of both enzymes are due to their differing affinities for hydrophilic and hydrophobic surfaces.

Acute Disease↗
PubMed1984

Cooperative transient trapping of photosystem II protons by the integral membrane portion (CF0) of chloroplast ATP-synthase after mild extraction of the four-subunit catalytic part (CF1).

The ATP-synthase in chloroplasts is built from two blocks, CF0, which is integral to the thylakoid membrane and which serves as a proton channel, and CF1, attached to CF0, which is catalytically active. This study is aimed at understanding proton conduction through CF0. By a mild procedure we extracted <10% of total CF1, predominantly the four-subunit CF1 without the delta subunit. Extracted chloroplasts were excited with short flashes of light and the time course of the transmembrane potential and of the pH changes in both phases was measured spectrophotometrically. Mild extraction of CF1 caused two effects. (i) Up to 50% of the protons rapidly released from water oxidation transiently escaped detection in the thylakoid interior. (ii) The initial extent of the transmembrane potential was decreased by some 10% (20-mus resolution). Protons that were not detected inside appeared in the external phase after having passed the thylakoid membrane. pH titrations of the transient loss of protons produced an extremely sharp transition (near pH 7.5) as if six protons were buffered in a strictly cooperative manner. These effects were reversed upon addition of N,N'-dicyclohexylcarbodiimide, which, among other actions, blocks the proton channel through CF0. We interpret these observations as follows. (i) CF0 incorporates proton binding groups, which can act in a hexacooperative way. These groups are located near the middle of the membrane. (ii) After extraction of CF1, protons produced during water oxidation have very rapid access to these groups, but they pass the full span of the membrane more slowly: buffering precedes conduction through CF0.

Journal Article↗
PubMed1984

Serum esterase activity in reactive systemic amyloidosis and its relation to amyloid A degrading activity.

Patients with reactive systemic amyloidosis have a reduced ability to degrade amyloid A protein fibrils in vitro. The amyloid A degrading activity in serum has been attributed to a neutral serine protease or proteases. Our results show that patients with reactive systemic (amyloid A) amyloidosis have low activities of two serum esterases, namely, arylesterase and paraoxonase, whereas the activity of a third esterase, cholinesterase, is normal. The combination of reduced arylesterase (less than 55 kU/L) plus reduced paraoxonase activity (less than 35 U/L) was found in 32% of patients with rheumatoid arthritis complicated by amyloidosis, but in only 5% of a control nonamyloid patient group, including patients with rheumatoid arthritis, liver disease, and hypoalbuminemia (p less than 0.001). A significant correlation between serum arylesterase and amyloid A degrading activity was found (patients with rheumatoid arthritis plus amyloidosis, n = 31, r = 0.51, p less than 0.01; all patients, n = 95, r = 0.34, p less than 0.001). Our results suggest that the amyloid A degrading activity may be closely related to the esterase activity in serum.

Adolescent↗
PubMed1983

Influence of colipase on the turbidimetric determination of pancreatic lipase catalytic activity.

The influence of colipase on the turbidimetric measurement of the catalytic activity of pure human pancreatic lipase (EC 3.1.1.3) and of sera from pancreatitis patients was studied. A deoxycholate-stabilized triolein emulsion served as substrate. It was found that the activity of the pure, colipase-free lipase is strongly inhibited by deoxycholate, and can be blocked completely if normal serum, pure human albumin, or the globulin fraction of normal serum is present. The inhibition by serum is competitive. This finding largely excludes the existence of a specific lipase inhibitor in human serum and explains the non-linear response of activity to the amount of serum added, a frequently observed problem with various turbidimetric lipase methods. A high molar excess of colipase (greater than 250-fold) completely abolishes the inhibition of lipase, irrespective of the inhibitory factor studied. Sera of pancreatitis patients, when measured turbidimetrically without addition of colipase, exhibit elevated lipase activity only if they contain colipase. However, the activity measured is not a function of the serum lipase concentration alone but of the molar ratio of colipase to lipase. Since this ratio varies considerably and is usually too low to ensure complete activation of lipase, erroneously low or even false negative results are obtained. For this reason it is strongly recommended that an excess of colipase is used in turbidimetric lipase assays. It therefore also appears important to study the influence of the serum colipase level on non-turbidimetric lipase methods.

Blood Proteins↗
PubMed1982

Detection of colipase in serum and urine of pancreatitis patients.

Colipase, like other pancreatic proteins, is liberated into the circulation in acute pancreatitis. Its concentration was measured in serum by a turbidimetric and in urine by a titrimetric method. The principle of both assays is based on the reactivation of bile acid inhibited, pure human pancreatic lipase by colipase. Whereas in healthy individuals colipase was found neither in serum nor urine (detection limit approximately 6.5 micrograms/1), a wide concentration range was observed in 29 patients with acute pancreatitis. Urine values varied between 3.8 and 7121 micrograms colipase/g creatinine; in serum levels up to 664 micrograms/1 were found. There was no correlation with serum lipase activity: On a molar basis, the ratio of serum colipase to serum lipase ranged between less than 0.04 and 2.14, but was below 1 in most sera. Colipase is rapidly removed from the circulation by glomerular filtration, its elimination rate from serum being more than twice as fast as that of lipase. This results in a constant decrease of the colipase/lipase ratio during the course of the disease. Probably determination of colipase is of no direct diagnostic value in pancreatic disorders, but our findings are of considerable significance for the measurement of serum lipase in the presence of bile acids, particularly with regard to turbidimetric assays. We conclude that lipase activity values obtained by these methods are mainly dependent on the degree of saturation of the enzyme with its cofactor and not on the true lipase concentration.

Amylases↗
PubMed1981

Rapid calcium release and proton uptake at the disk membrane of isolated cattle rod outer segments. 1. Stoichiometry of light-stimulated calcium release and proton uptake.

We reported a rapid, light-stimulated release of calcium from isolated rod outer segments that is apparent only when both the disk membrane and the plasma membrane are made permeable to calcium by adding the ionophore A23187 [Kaupp, U. B., Schnetkamp, P. P. M., & Junge, W. (1979) Biochim. Biophys. Acta 552, 390-403]. In this paper, we have investigated the light-sensitive diskal binding sites and the calcium release mechanism in their dependence on the pH and the presence of mono- and divalent cations, including calcium itself. We have observed now that several different rod outer segment preparations (i.e., rod outer segments with an intact plasma membrane, broken cells, and sonicated material) possess a similar dependence of their calcium release on the ionic conditions, however, only if manipulated in a way that gives access to the outer conditions of sites within disks (namely, ionophore added in the case of intact rod outer segments). Monovalent cations, at concentrations between 20 and 40 mM, suppress light-induced calcium release. Divalent and trivalent cations are more efficient inhibitors by 1-2 and 2-3 orders of magnitude, respectively. These results suggest that calcium release is controlled by an interfacial potential. The optimum pH for calcium release is pH 6.3, and virtually no release occurs beyond pH 4.5 and 9. The drop for acidic pH is attributed to the pH dependence of calcium binding to disk membranes, and the drop for alkaline pH is attributed to the pH dependence of the metarhodopsin I/metarhodopsin II transition and the light-stimulated proton uptake. In general, calcium release parallels calcium binding as a function of pH and calcium concentrations, although the release saturates at lower calcium concentrations ((KDapp = 5 microM) than would be expected from the amount of calcium bound (KD = 30-60 microM). The maximum stoichiometry is approximately 1 mol of calcium release per mol of rhodopsin bleached. Concomitant measurements of the light-stimulated uptake of protons by the disk membrane revealed a maximal stoichiometry of 2.8 mol of protons taken up per mol of rhodopsin bleached. We present an integrated description of light-stimulated calcium release, proton uptake, and changes of the interfacial potential at the disk membrane.

Animals↗