Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity.

As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.     

Analysis of peptides from animal and plant tissues

A new technique was developed for the analysis of peptide compositions of extracts from various animal and plant tissues. It involves the acidic extraction of a peptide fraction from the starting material and its precipitation with acetone, fractionation of peptides by ion-exchange chromatography by using a stepwise elution of fractions and detection by means of ninhydrin color reaction, and computer processing of the results. For the presentation of the results of analysis, chromatographic profiles and peptidograms were proposed. The results of analysis can be stored in a database and used for the creation of "generalized peptide portraits" and "differential peptide portraits" of the subjects investigated, which allow the identification of peptides characteristic of the subjects. The amount of peptide undergoing analysis ranged from 1 to 10 nmol.     

Acid hydrolase activity in tissues of mice after physical stress

• 1.1. The activities of β-glucuronidase and cathepsin D and the protein concentration were assayed from brain, kidney, liver, cardiac muscle and skeletal muscle (m. rectus femoris) samples from mice (Mus musculus) 1, 3, and 6 days after intermittent exhaustive (duration 100–145min) and submaximal prolonged (duration 9 hr) running on treadmill.
• 2.2. The activity of β-glucuronidase in skeletal muscle strongly increased being the highest 3 days after both exertions. Cathepsin D activity also slightly increased. In cardiac muscle β-glucuronidase activity was unaffected. Cathepsin D activity slightly increased 3 days after intermittent exhaustive exercise.
• 3.3. The specific activities of β-glucuronidase and cathepsin D in the liver increased 1 day after the both exertions. Simultaneously the protein concentration decreased. In the kidney β-glucuronidase activity and protein concentration were unaffected but cathepsin D activity decreased 1 day after intermittent exhaustive exercise.
• 4.4. In the brain protein concentration transiently decreased 3 days after the exertions. β-Glucuronidase activity transiently decreased 1 day after intermittent exercise thereafter increasing 6 days afterwards above the control level. Cathepsin D activity decreased 1 day after intermittent exercise but was unaffected after prolonged submaximal exercise.
• 5.5. Physical stress affected to varying extent the acid hydrolase activities in all organs studied.

     

Assay of superoxide dismutase activity in animal tissues

Convenient assays for superoxide dismutase have necessarily been of the indirect type. It was observed that among the different methods used for the assay of superoxide dismutase in rat liver homogenate, namely the xanthine-xanthine oxidase ferricytochromec, xanthine-xanthine oxidase nitroblue tetrazolium, and pyrogallol autoxidation methods, a modified pyrogallol autoxidation method appeared to be simple, rapid and reproducible. The xanthine-xanthine oxidase ferricytochromec method was applicable only to dialysed crude tissue homogenates. The xanthine-xanthine oxidase nitroblue tetrazolium method, either with sodium carbonate solution, pH 10.2, or potassium phosphate buffer, pH 7·8, was not applicable to rat liver homogenate even after extensive dialysis. Using the modified pyrogallol autoxidation method, data have been obtained for superoxide dismutase activity in different tissues of rat. The effect of age, including neonatal and postnatal development on the activity, as well as activity in normal and cancerous human tissues were also studied. The pyrogallol method has also been used for the assay of iron-containing superoxide dismutase inEscherichia coli and for the identification of superoxide dismutase on polyacrylamide gels after electrophoresis.     

A bioassay for insect deterrent compounds found in plant and animal tissues

A general field bioassay for detecting biologically active compounds in plants and insects has been developed and tested for efficacy and sensitivity. Methanolic extracts, in sucrose solution, of 20 plant and six caterpillar species were offered to the ponerine ant Paraponera clavata and the feeding preferences observed. The bioassay resulted in the detection of nine plant and three caterpillar species with ant-deterrent extracts, and 11 plant and three caterpillar species with neutral or attractant extracts. All of the plants showing ant-deterrent characteristics which had been chemically investigated in our laboratory, or for which chemical literature was available, contained secondary metabolites of known deterrence. Both naturally occurring and artificial differences in chemical concentrations could be detected using the bioassay. The method provides a means of screening plants and insects for compounds that are insect anti-feedants or that can modify insect behaviour.     

Photodynamic activity of cercosporin on plant tissues

Abstract Cercosporin, excited by incandescent light in the presence of oxygen, induced a loss of ions from the plant tissues tested. Silymarin, BHA (2(3)-tert-butyl-4-hydroxyanisole) and EDTA were effective in lowering the activity of cercosporin. Circumstantial evidence suggests that the toxic effect may be mediated by the singlet oxygen and free radical formation which induces a lipoperoxidative degradation of the polyunsaturated fatty acids of cell membranes.     

Peptide hydrolase activity in brain tissues during the early stages of post-hypothermia period

The acid peptidohydrolase activity in the homogenate, dissoluble and mitochondrial-lysosomal fractions of brain tissues of rats who have endured deep hypothermia was determined after their "active" warming for an hour and on the 1st, 2nd, 3d and 7th days after their self-warming. The "active" warming of rats who have endured deep hypothermia (19-20 degrees C) brings about the restoration of the acid peptidohydrolase activity in the subcellular brain tissue fractions. After self-warming the examined enzyme activity restores 7 days later. In the dynamics of the posthypothermic period a change in the acid peptide hydrolase distribution in fractions occurs on the 2nd-3d days.     

Ouabain-insensitive Na-ATPase activity in homogenates from different animal tissues.

1. Two Na(+)-stimulated ATPase activities were determined in gill homogenates from squid, shrimp and teleost fish; in kidney slice homogenates from teleost fish, bullfrog, toad, iguana, chicken, duck, rat, pig and cow, as well as in homogenates from rat small intestinal cells, brain cortex and liver slices. The two Na(+)-stimulated ATPase activities, the Na- and the Na,K-ATPase, showed a different behavior toward K+ and ouabain. 2. The ouabain-insensitive, K(+)-independent, Na-ATPase activity for all the studied homogenates was completely inhibited by 2 mM furosemide. 3. An increase in cell volume of the kidney, brain cortex and liver slice preparations, as well as of the rat small intestinal cells, produced a concomitant increase of the ouabain-insensitive Na-ATPase.