Enzymes In Tissue Samples Research Articles

Distribution and characterization of nitric oxide synthase in the nervous system of Triatoma infestans (Insecta: Heteroptera)

The biochemical characterization of nitric oxide synthase (NOS) and its distribution in the central nervous system (CNS) were studied in the heteropteran bug Triatoma infestans. NOS-like immunoreactivity was found in the brain, subesophageal ganglion, and thoracic ganglia by using immunocytochemistry. In the protocerebrum, NOS-immunoreactive (IR) somata were detected in the anterior, lateral, and posterior soma rinds. In the optic lobe, numerous immunostained somata were observed at the level of the first optic chiasma, around the lobula, and in the proximal optic lobe. In the deutocerebrum, NOS-IR perikarya were mainly observed in the lateral soma rind, surrounding the sensory glomeruli, and a few cell bodies were seen in association with the antennal mechanosensory and motor neuropil. No immunostaining could be detected in the antennal nerve. The subesophageal and prothoracic ganglia contained scattered immunostained cell bodies. NOS-IR somata were present in all the neuromeres of the posterior ganglion. Western blotting showed that a universal NOS antiserum recognized a band at 134 kDa, in agreement with the expected molecular weight of the protein. Analysis of the kinetics of nitric oxide production revealed a fully active enzyme in tissue samples of the CNS of T. infestans.

Evaluation of changes in the activity of proteolytic enzymes and their inhibitors in the processes that accompany the growth of gastric cancer

Many investigators have observed a correlation between the aggressiveness of malignant tumor growth and the levels of cysteine peptidases and their autogenous inhibitors. Cathepsins B and L are activated by pepsin in an acidic pH. This fact encouraged us to measure the activity of these enzymes in tissue samples of gastric cancer. We measured the activities of cathepsins B and L, and their precursors and inhibitors, in homogenates of tissue samples obtained from operations for gastric cancer. We compared the results for the homogenates of tissues from three different sites: the tumor center, the zone of cancer invasion (border of the tumor), and healthy tissue. The highest activities of free cysteine peptidases and those in complexes with their inhibitors, as well as their precursors, were observed in the border of the tumor, while the activities in healthy tissue were significantly lower. The local activities of cysteine peptidases and their inhibitors reflect the topographical differences between the center of the tumor, the zone of invasion, and healthy tissues in gastric cancer patients. In addition, the results for the pattern of changes in the activity of cysteine peptidases according to the degree of tissue infiltration were not dependent on the method of measurement (colorimetry vs spectrofluorometry).

Stability of diacylglycerol acyltransferase in dehydrated bovine muscle tissue

Meaningful estimates of diacylglycerol acyltransferase (EC 2.3.1.20) activity in different tissue samples require effective, unbiased methods of sample storage. Samples of the pars costalis diaphragmatis muscle (skirt muscle of the diaphragm) were obtained from 18- to 20-month-old cattle and assayed for microsomal protein content and diacylglycerol acyltransferase activity after having been stored under various conditions as dissected tissue or microsomes prepared from dissected tissue. There was relative enrichment of diacylglycerol acyltransferase specific activity ( p<0.05) when samples prepared from the pars costalis diaphragmatis muscle were dehydrated and stored for 2 weeks, as compared to the control condition (in which the microsome fraction was prepared from fresh pars costalis diaphragmatis muscle and assayed immediately). The results suggested that dehydration was an effective method of storage for bovine muscle samples destined for estimation of the microsomal diacylglycerol acyltransferase activity. The dehydration approach for preparing samples for analysis of diacylglycerol acyltransferase activity might also prove useful to investigators who are interested in obtaining reliable estimates of the activity of other enzymes in tissue samples.

A protein activator of galactosylceramide β-galactosidase

A heat-stable protein was isolated from the spleen of a patient with Gaucher's disease. This protein will activate glucosylceramide β-glucosidase activity (Ho, M.W. and O'Brien J.S. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 2810–2813). When the specificity of this activator was tested using other enzymes and substrates, it was found to activate galactosylceramide β-galactosidase activity and sphingomyelinase but not GM1 β-galactosidase or sulfatide sulfatase. The ability to stimulate galactosylceramide β-galactosidase was optimum at pH 4.6 in the presence of pure phosphatidylserine or other acidic lipids such as sulfatide and phosphatidylinositol. The partially purified activator protein could stimulate galactosylceramide β-galactosidase activity in brain, liver, leukocytes and cultured fibroblasts. It was not able to stimulate the activity of this enzyme in tissue samples from patients with Krabbe's disease, demonstrating that it was acting on galactosylceramide β-galactosidase and not GM1 β-galactosidase. It was slowly denatured by treatment with Pronase, reaching 16% of starting levels after 24 h at 50° C. Attempts to separate the abilities of this activator preparation to stimulate several lysosomal hydrolases by column chromatography were not successful.

N-Acetyl-Glucosaminidase Activity during Limb Regeneration in the Adult Newt

N-Acetyl-glucosaminidase activity was measured during the first 25 days of limb regeneration. It was found that the enzyme is present in the normal limb. Following amputation a significant drop was obtained at day 3. A significant increase in enzyme activity was found at day 5 followed by a second drop by day 10. For days 12–15 a second peak of enzyme activity was detected, followed by a third drop; by day 25, normal levels of enzyme activity were detected. Histochemical localization of the enzyme in tissue samples showing enzyme activity as detected biochemically (days 5 and 17 of regeneration) gave negative results. However, enzyme activity was found in the incubation medium, indicating that the enzyme is released from the cells. The peaks of enzyme activity coincide with the stages of limb regeneration where a high degree of tissue demolition and cell lysis occurs. The latter are important events in the regeneration process, cell dedifferentiation, and blastema formation.

Fatty Metaplasia of the Filum Terminale of the Frog

THE following observations were made during a search for preparations which would permit assays of various enzymes in tissue samples containing glial cells only. Such an opportunity seemed to be provided by the filum terminale which is commonly considered a glial remnant of the fœtal sacral cord1. Tobias, Clark, and Gerard2 measured the respiration of the filum terminale of the cat and concluded that glial tissue had a rather high rate of respiration, comparable to that of neurones. The filum terminale of Rana catesbiena was used in our work because of its unusual size. A smaller material of 8 specimens of Rana pipiens was also examined with the same results.