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S E Brolin

Publications and source records attributed to S E Brolin.

At least 37 records · Page 2Linked to original sources
PubMed1979

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.

Adenylate Kinase↗
PubMed1978

Adenylate kinase activity in various organs and tissues of mice with the obese-hyperglycemic syndrome (gene symbol Ob/Ob).

The activities of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in lyophilized cryostat sections of various organs from mice with the obese-hyperglycemic syndrome (gene symbol ob/ob) were measured fluorometrically. Skeletal and heart muscle tissues had the highest enzyme activities, i.e. 258 and 155 mmoles substrate converted per kg dry weight and per hr (MKH), respectively. Pancreatic islet specimens, which were composed mainly of B-cells, possessed the highest activitty of all parenchymatous cells studied, with 18 MKH as compared to 16 MKH for the cortical tubules of the kidney and 7 and 6 for the exocrine pancreas and liver cells, respectively. The granular layer of the cerebellum had enzyme activity of 31 MKH. The adenylate kinase activity in the B-cells was apparently not influenced by hyperglycemia since it was similar both in obese-hyperglycemic mice of different ages and in lean mice, although the blood sugar concentrations in such animals are much different.

Adenylate Kinase↗
PubMed1978

Fluorometric microassays of adenylate kinase, an enzyme important in energy metabolism.

The adenylate kinase system offers a mechanism for the rapid provision of energy by catalysing the production of ATP from ADP. Fluormetric micromethods were developed for determination of the activity of this enzyme using either formation of ADP or ATP, in each case measured by coupling to suitable dehydrogenase reactions. Both procedures yielded results in good agreement, but when ADP formation was measured an interfering phosphatase splitting of ATP had to be corrected for. Therefore, ADP was preferred as the substrate and its conversion to ATP was determined in a coupled hexokinase-glucose-6-phosphate dehydrogenase reaction yielding stoichiometric amounts of NADPH which were measured by the native fluorescence of this form of the nucleotide. The sensitivity and reproducibility of our micro-method permitted assay of small samples (50-500 ng) such as a layer of cerebellar cortical nerve cells and of insulin producing cells from the islets of Langerhans. Although not reaching the high values in muscle, these cells showed significantly higher activities than parenchymatous cells from the liver and the exocrine pancreas. The sensitivity attained is more than required for assay of clinical fine needle biopsies and is quite satisfactory for detection and estimation of adenylate kinase contaminants in enzyme preparations.

Adenosine Diphosphate↗
PubMed1977

Microassay with the NADH-induced light reaction, technique improved by means of purified enzymes from Achromobacter fischeri.

Purification of a commercial preparation of Achromobacter fischeri was carried out by agarose-suspension electrophoresis and by molecular-sieve chromatography. Both the luciferase and an oxidoreductase, catalyzing reduction of FMN with NADH, were obtained in more than one form. Flavins, liable to interfere with the light production in analytical applications, were present in amounts worthy of consideration, but seem to be firmly bound to protein. The major quantity was found in the enzymatically inactive fractions. In free zone electrophoresis of the main luciferase component, the mobility of the zone containing enzyme activity was calculated to -4.0 X 10(-5) cm2 sec-1 V-1 at 12 degrees C. Fractions of the two enzymes were separated and mixed in different proportions to study how the intensity and time course of NADH-induced light emission can be modified. These experiments disclosed how reaction mixtures will have to be composed in appropriate photokinetic assays, using NADH as measurable product. A regenerating system based on the purified fractions is described. Instead of the light flash, following the consumption of NADH, the light is emitted on a well maintained level, permitting assays with a less elaborate equipment than the one required for the recording of fast reactions.

Chemistry Techniques, Analytical↗
PubMed1977

Assay of flavin nucleotides in pancreatic islets by a differential fluorimetric technique.

Differences in fluorescence intensity between FMN and FAD at high and low pH values were utilized to determine the concentration of each nucleotide. Amounts down to 1 pmol were measurable. The FMN and FAD contents of pancreatic islets of obese-hyperglycaemic mice (gene symbol ob/ob) were compared with those of the exocrine pancreas, the liver and heart muscle. As expected, the FAD content of each tissue was greater than that of FMN. The contents of both nucleotides were significantly higher in the islets than in the exocrine part of the pancreas, but lower than in the heart muscle and liver. Possible relationships between the flavin content of the islets and their capacity for oxidative phosphorylation are briefly discussed.

Animals↗
PubMed1977

Chemiluminescence microanalysis of substrates and enzymes.

Extracts from bioluminescent organisms are increasingly used for analysis of small amounts of substrates and enzymes. The light emission is in some organisms related to the conversion of substrates and cofactors of central metabolic importance. Extracts from such organisms are particularly valuable for analytical applications. This is quite obvious in the firefly where the energy, required for light production, is derived from ATP and in a couple of strains of luminescent bacteria where reduced pyridine nucleotides through reduction of flavine mononucleotide is utilized in the light reaction. It deserved to be noted that many biochemical reactions can be coupled more or less directly to the conversion of ATP, NAD(H), NADP(H) and FMN(H), thus providing the basis for a great variety of analyses. Special kinds of bioluminescent reactions are also of considerable interest, as for instance the relationship between "active sulphate" and PAP, which participate in the formation of light in the sea pansy (Renilla reniformis). High sensitivities are often reached in chemiluminescence analysis making the technique suitable for samples composed of a small number of cells. How bioluminescence has been employed in these kinds of microanalyses is examplified in studies of nucleotides, metabolites and enzymes with low activities.

Adenosine Triphosphate↗
PubMed1977

Hydroxyacyl CoA dehydrogenase, an enzyme important in fat metabolism in different cell types in the islets of Langerhans.

The pancreatic islets show a remarkably high activity of L-3-hydroxy-acyl CoA dehydrogenase, an enzyme which operates in the fatty acid cycle by catalyzing the NAD+ oxidation of some of the degradation products. In order to study the distribution pattern of its activity within the islets, samples with different relative contents of A1-, A2- and B-cells were prepared and analyzed. The results show that it is unlikely that either the A1-cells or the enzymatically well equipped A2-cells contribute to the high activity values of the islets. In contrast, the experiments indicated that the high activity was due to the B-cells. After 72 hours starvation, leading to an increase in the serum free fatty acids, there was no change in the activity of the A2-cells, while the B-cells, however, showed a significant but moderate decrease in their activity. It is concluded that the B-cells are enzymatically equipped for the oxidation of fatty acid degradation products even in situations with diminished activity such as occurs during a decrease of the mitochondrial assembly.

3-Hydroxyacyl CoA Dehydrogenases↗