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H Winkler

Publications and source records attributed to H Winkler.

At least 271 records · Page 15Linked to original sources
PubMed1983

[Formation of plastein gels from peptic digests of field bean protein isolates].

Peptic partial hydrolysates of a different hydrolysis degree were prepared from horse bean protein isolate and used for the plastein reaction with thermitase. Whilst unsoluble products were formed from partial hydrolysates of a hydrolysis degree above 60%, plastein gel could be obtained from a less intensely split basic material. By gel chromatography and by determination of the percentage which was unsoluble in 10% trichloroacetic acid, a proteolytic degradation could be made proof on the conditions of the plastein reaction. On addition of DL-methionine ethylesters to the partial hydrolysate an increasing incorporation of methionine into the plastein could be observed with increasing hydrolysis degree.

Amino Acids↗
PubMed1983

Is lysolecithin an in vivo constituent of chromaffin granules?

It was recently claimed that lysolecithin (lysophosphatidylcholine) in chromaffin granules is a post-mortem artefact. We have, therefore, determined catecholamine/lysolecithin ratios in adrenal tissues and isolated chromaffin granules. In rat adrenals and bovine medulla the ratios in both tissues and granules were similar. This indicates that even in rapidly frozen rat adrenal glands, sufficient lysolecithin is present in the total tissue to account for its presence in isolated organelles. Owing to the high cortex/medulla ratios such studies cannot be performed with guinea pig or rabbit adrenals. However, isolated chromaffin granules from guinea pig, in contrast to a previous study, do contain lysolecithin. We conclude that lysolecithin is an in vivo constituent of chromaffin granules of all species so far investigated.

Adrenal Medulla↗
PubMed1983

On the temperature- and salt-dependent conformation change in human erythrocyte pyruvate kinase.

The influence of temperature, K+, Mg2+ and fructose 1,6-bisphosphate on human red cell pyruvate kinase was investigated. Kinetic measurements between 4 degrees C and 43 degrees C revealed a remarkable influence of the temperature on the allosteric behaviour of the enzyme. Below a transition region between 15 degrees C and 20 degrees C (as obtained from an Arrhenius plot) the enzyme shows non-cooperative behaviour, as can be deduced from Michaelis-Menten, Hill and Scatchard plots. At temperatures above 20 degrees C cooperativity increases with rising temperature. This effect becomes even more pronounced at higher temperatures upon addition of increasing amounts of K+ and Mg2+ accompanied by a slight decrease of the reaction velocity. Fructose 1,6-bisphosphate, however, abolishes cooperativity at every temperature and salt concentration measured. Difficulties which arise in evaluating the correct values of V, Km and the Hill coefficient nH with cooperative systems are met by using a computer program of Wieker, Johannes and Hess, especially designed for the determination of kinetic parameters obtained from sigmoidal steady-state kinetics.

Erythrocytes↗
PubMed1983

Membrane events in adrenal chromaffin cells during exocytosis: a freeze-etching analysis after rapid cryofixation.

Catecholamine-storing chromaffin cells, isolated from bovine adrenal medullae by collagenase digestion, were stimulated with carbachol at 37 degrees C; aliquots for controls were kept at 37 degrees C: Starting from this temperature, cells were ultrarapidly frozen with the use of a sandwich-propane-jet procedure and freeze-fractured. The replicas were analysed quantitatively for exocytotic activity: After stimulation the cell membrane displayed a significant increase of exo-endocytotic openings varying in size from 20 to 300 nm. The number of openings increased in parallel to the catecholamine output. At no stage could a clearing of membrane-intercalated particles (MIPs) be observed. Openings of all size classes were etchable. Results from the PF-face were comparable with those of the EF-face. We conclude that (i) exocytosis in isolated chromaffin cells starts as a focal event; the smallest possible stages are about 10 nm in size, (ii) fusion proceeds without previous rearrangement of MIPs, and (iii) the opening starts without formation of a diaphragm.

Adrenal Medulla↗
PubMed1983

Erythrocyte pyruvate kinase deficiency. A kinetic study of the membrane-localised and cytoplasmatic enzyme from six patients.

Investigations on the activity and the kinetic behavior of red cell pyruvate kinase in the erythrocyte membrane and hemolysate of 6 patients with pyruvate kinase (PK) deficiency are presented and compared with those of normal individuals. Hemolysate: The PK in the hemolysate of normal individuals showed the well-known allosteric properties, reflected by sigmoidal reaction kinetics (Hill-coefficient greater than 1). In contrast to these findings the enzymes of 4 of the patients which had not been splenectomised and which were all in satisfactory condition showed between 15 and 35% of the PK activity of normal individuals in the hemolysates, whereas in the case of the 2 splenectomised patients only 5 and 15% of this activity could be measured. All of the patients' enzymes showed non allosteric (hyperbolic) reaction kinetics (Hill-coefficient approximately 1.0). Red cell membranes: Our studies confirmed earlier reports that not only the hemolysate of the patients, but also their red cell membranes ('ghosts') exhibit PK activity. In normal individuals the activity of the membrane-localised PK is about 5% of the cytoplasmatic one. In case of the membrane-localised enzyme of all patients--irrespective if they were splenectomised or not--the activity is decreased to 30-50%, compared to the activity of ghosts of the control persons. Reaction kinetic studies on the PK isolated from red cell membranes from normal individuals as well as from all 6 patients exhibited hyperbolic Michaelis-Menten kinetics with Hill coefficients around 1.0.

Adolescent↗
PubMed1982

High-performance liquid chromatographic method for the quantitative analysis of the aryloxypropanolamines propranolol, metoprolol and atenolol in plasma and tissue.

A simple and rapid high-performance liquid chromatographic method is described for the quantitative analysis of three beta-receptor blocking drugs of similar molecular structure (aryloxypropanolamines, AOPAs) but with different polarities. the method consists of extraction, reversed-phase ion-pair chromatography and fluorometric detection, whereby slight modifications in these parameters allow analysis of the different AOPAs in a similar way. The method was used to determine concentrations of propranolol, metoprolol and atenolol in plasma and various organs of the rat. In samples of 1 ml of plasma and 0.5--1.7 g of tissue, drug concentrations of at least 2 ng/ml and 5 ng/g, respectively, can be measured. Extension of the method to other AOPAs is possible.

Animals↗
PubMed1982

The topography of gangliosides in the membrane of the chromaffin granule of bovine adrenal medulla.

We have studied the topography of the gangliosides of the adrenal chromaffin granules by using neuraminidase to remove sialic acid from membrane gangliosides of intact and ruptured chromaffin granules. Residual sialic acid was then measured to compare the availability of gangliosides on the outer and inner surfaces of the membrane. Measurement of protein sialic acid served as a control since these residues are known to be on the inner surface of the membrane. Prolonged digestion of broken membranes showed that maximally 75% of both lipid and protein-bound sialic acid residues are available to neuraminidase. Prolonged digestion of intact granules produced no measureable loss of sialic acid from either protein or lipid fractions. Comparison of the thin-layer chromatograms of gangliosides extracted from digested and undigested membranes showed no preferential digestion of any component. We conclude that at least 75% of the gangliosides are on the inner leaflet of the membrane and suggest that all of the gangliosides are so located.

Adrenal Medulla↗
PubMed1982

Similarity of secretory protein I from parathyroid gland to chromogranin A from adrenal medulla.

We have compared the amino acid and carbohydrate compositions, partial amino acid sequences, immunological crossreactivity, and physical properties of secretory protein I of the parathyroid gland and chromogranin A of adrenal gland. This comparison indicates that these proteins are similar molecules. Because secretory protein I is present in secretory granules containing parathormone and is cosecreted with the hormone, and because chromogranin A is contained within chromaffin granules and, likewise, is secreted with the catecholamines, the present observations raise the possibility that this class of protein plays a general role in hormone secretion or storage mechanisms.

Adrenal Medulla↗
PubMed1982

Characterization of catecholamine-storage organelles in transplantable phaeochromocytoma and adrenal glands of rats.

The properties of the catecholamine-storing organelles from transplantable rat phaeochromocytoma and rat adrenal glands were compared by density gradient centrifugation. It was shown that tumour granules are more heterogeneous and less dense than adrenal granules. Both granule preparations can take up catecholamines and nucleotides by a process driven by an electrochemical proton gradient. Dopamine beta-hydroxylase and glycoprotein III were analysed by immunological techniques. Glycoprotein III was shown to be a specific component of chromaffin granules. Tumour tissue (average weight 700 mg) contains amounts of these antigens comparable to those in 210 adrenals. The biosynthesis of granules in the tumour apparently occurs at a low rate, making turnover studies difficult. The transplantable rat phaeochromocytoma is very useful for studies on the uptake properties and the immunological characteristics of rat catecholamine storage granules because on tumour provides an amount of material that could otherwise be obtained only from a large number of adrenal glands.

Adenosine Triphosphate↗
PubMed1982

Dopamine beta-hydroxylase and other glycoproteins from the soluble content and the membranes of adrenal chromaffin granules: isolation and carbohydrate analysis.

Chromogranin A and two other proteins (A1 and A2) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N-acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N-acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine beta-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine beta-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins.

Adrenal Medulla↗
PubMed1982

Adrenal chromaffin granules: evidence for an ultrastructural equivalent of the proton-pumping ATPase.

Adrenal chromaffin granules are known to possess an F1-ATPase which according to biochemical criteria is very similar to the mitochondrial one. To find a morphological equivalent for this enzyme chromaffin granules from bovine adrenal medullar were subjected to negative staining and freeze-etching. With both methods globular particles of 8 to 9 min diameter could be demonstrated on the surface of these organelles. A single granule possessed on average 22 particles. In negative staining the particles appeared separated from the membrane by a stalk of 8 nm. This typical morphological appearance was independent from a great variety of experimental procedures. After freeze-etching the particles were closely apposed to the membrane without any evidence for an interposed stalk. Pretreatment of chromaffin granules with pronase or trypsin led to a time dependent disappearance of the surface particles. In negative staining the stalked of chromaffin granules were found to be very similar in structure and size to those of mitochondria which have already been identified as F1-complexes. Based on this observation and other lines of evidence we suggest that the stalk particles found on the surface of chromaffin granules represent the F1-complex of the proton-pumping ATPase of these organelles.

Adenosine Triphosphatases↗
PubMed1980

Red cell pyruvate kinase deficiency: an optimised assay.

As a result of using a variety of assay techniques, rather different values for the rate of the pyruvate kinase (PK) reaction were obtained. Kinetic measurements performed with red cell PK using the stopped-flow as well as the "classical" spectrophotometric method demonstrated that only the initiation of the reaction with ADP yields reproducible results in the standard assay. Moreover, the influence of substrate concentrations, activator concentrations, ionic strength, buffer system, temperature and duration of preincubation were thoroughly investigated and were shown to play an important role. As a consequence of our kinetic measurements, a modified optimised assay composition together with new normal values for erythrocyte PK activity in haemolysate are presented. For better characterisation and comparability of PK variants, we suggest the use of the optimised assay composition described and that the reaction be initiated with ADP.

Adenosine Diphosphate↗