Ambjörn Ågren

Photokinetic assay of pyruvate in the islets of Langerhans using bacterial luciferase.

A method is described determining the pyruvate content in 1–10 μg of lyophilized tissue. This represents a range of weight much below that of fine-needle biopsies. NAD+, generated through the enzymatic conversion of pyruvate to lactate, is reduced to NADH which is then assayed by its ability to induce light emission with luciferase extracts of Achromobacter fischerii. The sensitivity of the method is sufficient to determine 0.5 pmol of pyruvate. Values obtained by this method in lyophilized liver agree well with those obtained by spectrophotometric techniques. In applications of the method, it was found that the islet concentration of pyruvate was greater than in the exocrine pancreas but smaller than in the liver. The pyruvate content in tissues of the obese hyperglycemic mice was greater than in their lean litter mates, which may be explained by a more rapid rate of glucose utilization due to their hyperglycemia.

MORPHOLOGY AND ENZYME ACTIVITIES OF THE RETINAL CAPILLARIES IN STREPTOZOTOCIN-DIABETIC MICE

Male lean mire belonging to the obese‐hyperglycemic strain were made diabetic by intravenous injection of streptozotocin. The retinal capillary bed freed by trypsin digestion was studied with regard to morphology and the activity of some enzymes. There was a significant increase in the ratio between the endothelial and mural cells which was interpreted as indicating mural pericyte disappearance. The activities of adenylate kinase, aspartate‐amino‐transferase and hydroxyacyl‐CoA‐dehydrogenase in the retinal vessels of the diabetic animal were significantly higher than in vessels from the control animals. No differences were found in the activities of glucose‐6‐phosphate dehydrogenase, glutathione reductase and phosphofructokinase between the two animal groups.

Morphology and enzyme activities of the retinal capillaries in mice with the obese-hyperglycaemic syndrome (gene symbol ob).

The retinal capillary bed from 67 obese‐hyperglycaemic mice and 64 lean litter mates was isolated by trypsin digestion and investigated with respect to structure and enzyme activities. There was no significant difference in the ratio between numbers of endothelial and mural cells. The capillary walls did not show any obvious structural differences and microaneurysms were not observed. The retinal vessels from the obese‐hyperglycaemic mice, however, displayed significantly higher activities of the enzymes hydroxyacyl‐CoA‐dehydrogenase, asparate aminotransferase (ASAT) and adenylate kinase than their lean litter mates. The activities of glutathione reductase, glucose‐6‐phosphate dehydrogenase (G‐6‐PDH) and phosphofructokinase were similar in the two experimental groups. It is suggested that the present data reflect early metabolic disturbances related to diabetic retinopathy.

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.

The analytical use of lactate dehydrogenase entrapped in microparticles

Abstract Lactate dehydrogenase (LDH) was immobilized in microparticles of polyacrylamide. The particles were prepared by polymerization of acrylamide and bisacrylamide in an emulsion of water in toluene-chloroform. The polyacrylamide gel formed is macroporous and allows rapid equilibrium between the particles and the environment to take place. The enzyme was shown to be active by reducing pyruvate. The LDH-particles can conveniently be used in the analysis of picomole amounts of lactate or pyruvate.

Application of chemiluminescent probes in investigating lysosomal sensitivity to superoxide verus suspected radical scavengers

The role of the superoxide anion radical (O2(-).) relative to catalytic/inhibitory substances in lysosomes is poorly understood. Cultured glial cells sequestered endocytotically two probes that were site-specific to the lysosome vacuome and sensitive to radical activities. The Sepharose-4B-isoluminol probe emitted chemiluminescent light in proportion to externally injected O2(-). and was inhibited or catalysed by various radical scavengers, transition metals and other substances which may affect lysosomal metabolism and lipofuscin formation. We conclude that lysosomal radical activities may be inhibited by butylated hydroxytoluene, hydrocortisone, ACF, RNA, alpha-tocopherol, and, in special circumstances, with fully metabolized iron. Choline and oxidized copper and iron cations in overload concentrations in incubated freshly in lysosomes may catalyse radical activities, and they may be important factors in lipofuscin formation and its role in aging.

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.

A new method that investigates superoxide versus respiration endomitochondrially with a site-specific chemiluminescent probe

Abstract Metabolically active rat-liver mitochondria impregnated with a site-chemiluminescent probe emitted steady-state light generated by endomitochondrial superoxide and adducts. Endomotichondrial pH was estimated at 8.1±0.2 (7.8–8.3). The weakly basic environmental of the inner mitochondrial matrix is reasonably the result of respiratory-chain superoxide radicals reacting with water and producing hydroxyl anions.

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.

Potentialities of Bioluminescence Analyses in Research on the Pancreatic Islets

Progress in bioluminescence assay permits not only determinations of nucleotide and substrate concentrations, but also estimation of concentration shifts. The analyses can be extended to comprise Ca2+ since the Aequorea system is sensitive enough for applications in islet research. By connecting the bioluminometer to a microprocessor with a suitable readout device, it is possible to collect and evaluate large amounts of data which may be required in studies of concentration shifts. Thus, blanks, samples and standards can be processed completely within short time periods so that the light-yielding solutions remain stable.