Quantitative Histochemical Procedures Research Articles

SDH and actomyosin ATPase activities of different fiber types in rat diaphragm muscle.

In the rat diaphragm muscle, the histochemical classification of type I, IIa, IIb, or IIx fibers was correlated with myosin heavy chain (MHC) immunoreactivity. Expression of MHC isoforms in single dissected fibers was also assessed electrophoretically. Most fibers (approximately 86%) expressed a single MHC isoform, and when present, coexpression of MHC-2X and MHC-2B isoforms was most prevalent. Type I and IIa fibers were the smallest, type IIb fibers were the largest, and type IIx fibers were intermediate. Succinate dehydrogenase (SDH) and calcium-activated myosin adenosinetriphosphatase (actomyosin ATPase) activities were measured with quantitative histochemical procedures. Type I and IIa fibers had the highest SDH activities, followed in rank order by type IIx and IIb fibers. Type I fibers had the lowest actomyosin ATPase activity, followed in rank order by type IIa, IIx, and IIb fibers. Across all fibers, there was an inverse relationship between fiber SDH activity and cross-sectional area and a positive correlation between fiber actomyosin ATPase activity and cross-sectional area. The SDH and actomyosin ATPase activities of muscle fibers were also inversely correlated. These phenotypic differences in SDH and ATPase activities may be important in determining the contractile and fatigue properties of different fiber types in the rat diaphragm muscle.

Lessened malate inhibition of guard-cell phospho enolpyruvate carboxylase velocity during stomatal opening

Leaflets of Vicia faba with closed stomata or with opening stomata were freeze-dried. Excised guard-cell pairs were assayed individually under suboptimal conditions (pH 7.1 and subsaturating substrate) for phospho enolpyruvate carboxylase (PEPC; EC 4.1.1.31) using quantitative histochemical procedures. l-Malate, 400 μM, significantly inhibited guard-cell PEPC activity of closed stomata but not that of opening stomata. We postulate that the lessened sensitivity of guard-cell PEPC activity to malate inhibition is an important regulatory feature of stomatal opening, which is associated with malate accumulation.

Glucokinase in B-cell-depleted islets of Langerhans.

Glucose phosphorylation was studied in B-cell-enriched or in B-cell-depleted pancreatic islets from normal or streptozotocin-diabetic rats, respectively, using quantitative histochemical procedures. The data indicate that B-cell-enriched preparations from normal animals and whole islets from normals, diabetics, and insulin-treated diabetic animals have comparable glucokinase activities. Average maximum velocities were (mmol/kg dry tissue/hr) 134.1 +/- 7.3 for whole islets and 125.6 +/- 10.7 for the B-cell-enriched preparations from normal rats, 143.1 +/- 13.6 for B-cell-depleted islets from diabetic rats, and 124.4 +/- 10.7 for B-cell-depleted islets from insulin-treated diabetic animals. The Kmax for glucose of the enzyme in islets from untreated diabetic rats was 16 mM, comparable to the Kmax found for glucokinase from normal rat islets. Mannoheptulose, previously shown to be a competitive inhibitor of glucokinase from liver and normal islets, also inhibited glucokinase in B-cell-depleted islets from diabetic rats. The data indicate that glucokinase is not selectively located in the B-cell, as was previously assumed, but is also found in A- and/or D-cells of diabetic rats. This observation raises significant questions about the functional role of islet glucokinase under control and diabetic conditions.

Radiometric oil well assay for glucokinase in microscopic structures

Glucokinase (ATP: d-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet β-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP: d-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H 2O from d-[2- 3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase ( d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic β-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the K m and the Hill number) in microscopic samples of tissue.

Microchemical mapping studies of neurotransmitter chemistry in the cochlear nucleus

Quantitative histochemical procedures [O. H. Lowry and J. V. Passonneau, A Flexible System of Enzymatic Analysis (Academic, New York, 1972), pp. 219–260] have been applied together with a mapping approach to study the distributions of neurotransmitter systems in the cochlear nucleus. The methods involve quick‐freezing of tissue, followed by frozen sectioning, freeze‐drying, dissection of sections with microknives, weighing of samples on microbalances, and direct chemical assays in microliter volumes. Structures as small as 20 μM per edge can be chemically analyzed by these methods, and the histological locations of all samples are recorded, allowing direct comparisons with anatomical and physiological data for the same locations. The distributions of proposed transmitter amino acids and of the enzymes of acetylcholine metabolism have been mapped in the cochlear nuclei of rats and cats. The effects on these distributions of transections of anatomical pathways are being studied. Results so far suggest that, in the rat cochlear nucleus, most cholinergic synapses may be related to centrifugal, or feedback, pathways to the nucleus.

Distribution of Alkaline Phosphatase in Pollen Grains and Pollen Tubes of Calotropis procera

Summary Using qualitative (Gomori-Takamatsu and Naphthol phosphate procedures) and quantitative histochemical procedures, the distribution of alkaline phosphatase in pollen grains and pollen tubes of Calotropis procera R. Br. is demonstrated. Role of this enzyme in permeability, energy transfer reactions and intercellular transport mechanism is discussed.