scholarly journals LOW TEMPERATURE EMBEDDING IN GLYCOL METHACRYLATE FOR ENZYME HISTOCHEMISTRY IN PLANT AND ANIMAL TISSUES

1972 ◽  
Vol 20 (12) ◽  
pp. 986-990 ◽  
Author(s):  
ANNE E. ASHFORD ◽  
WILLIAM G. ALLAWAY ◽  
MARGARET E. MCCULLY
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Enzyme Histochemistry ◽  
Animal Tissues ◽  
Glycol Methacrylate

A method is described by which enzyme activity can be retained in plant and animal tissues embedded in glycol methacrylate at low temperature.

scholarly journals Enzyme histochemistry on freeze-dried, resin-embedded tissue.

1989 ◽  
Vol 37 (5) ◽  
pp. 643-652 ◽  
Author(s):  
G I Murray ◽  
M D Burke ◽  
S W Ewen
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Enzyme Histochemistry ◽  
Aqueous Media ◽  
Histochemical Demonstration ◽  
Glycol Methacrylate ◽  
Tissue Sections ◽  
Freeze Dried ◽  
Wide Range ◽  
Tissue Specimens

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

scholarly journals Enzyme histochemistry on freeze-substituted glycol methacrylate-embedded tissue.

1990 ◽  
Vol 38 (1) ◽  
pp. 95-101 ◽  
Author(s):  
G I Murray ◽  
S W Ewen
Keyword(s):  
Low Temperature ◽  
Enzyme Histochemistry ◽  
Aqueous Media ◽  
Freeze Substitution ◽  
Histochemical Demonstration ◽  
Enzyme Localization ◽  
Glycol Methacrylate ◽  
Tissue Sections ◽  
Wide Range ◽  
Tissue Specimens

We developed a method for histochemical demonstration of a wide range of enzymes in freeze-substituted glycol methacrylate-embedded tissue. Tissue specimens were freeze-substituted in acetone and then embedded at low temperature in glycol methacrylate resin. All enzymes studied (oxidoreductases, hydrolases) were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-substitution combined with low-temperature glycol methacrylate embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, maintenance of enzyme activity, and excellent tissue morphology.

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

1978 ◽  
Vol 26 (10) ◽  
pp. 772-781 ◽  
Author(s):  
W D Klohs ◽  
C W Goff ◽  
R J Bernacki
Keyword(s):  
Enzyme Activity ◽  
Lead Nitrate ◽  
Initial Step ◽  
Lead Ions ◽  
Fixation Time ◽  
Animal Tissues ◽  
Root Tips ◽  
Cytochemical Localization ◽  
Onion Root ◽  
L1210 Cells

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.

scholarly journals GENETIC EVIDENCE FOR INTERACTION BETWEEN NONHOMOLOGOUS PROTEINS IN YEAST AND A CASE OF SUPPRESSION AT THE HIS1 LOCUS

Genetics ◽  
1980 ◽  
Vol 94 (2) ◽  
pp. 327-339 ◽  
Author(s):  
Richard Snow
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Enzymatic Activity ◽  
Temperature Effect ◽  
Conformational Change ◽  
Minimal Medium ◽  
Genetic Evidence ◽  
Structural Genes ◽  
Wild Type ◽  
Is Strain

ABSTRACT The HIS1 and THR4 loci are the structural genes for phosphoribosyl-ATP pyrophosphorylase and threonine synthetase, respectively. The allele his1-IS has no enzyme activity at 30", but does have activity at 15" provided the cell contains the wild-type THR4 allele or a suppressing allele at another locus, designated SUP(his1-1S). Under these conditions, cells with the hisl-IS mutation are capable of growth on minimal medium at 15". Three kinds of reversions of a hisl-IS thr4 sup(his1-IS) strain to histidine prototrophy have been obtained: (1) his1-IS locus reversions to HIS1 that restore growth without added histidine at 30", (2)  thr4 reversions to THR4 that simultaneously eliminate the requirement for threonine and restore the low-temperature effect on the his1-IS allele, and (3)mutations from sup to SUP. The SUP allele is not an ochre suppressor, and it is not linked to either HISI, THR4 or a centromere. It may represent a missense suppressor. I t is proposed that the effect ofTHR4 is caused by aggregation of the wild-type threonine synthetase with defective his1-IS monomers, causing a favorable conformational change in the histidine protein that restores limited enzymatic activity. This can be regarded as a case of complementation between nonhomologous proteins.

Parallel Intralysosomal Amyloid Fibrils, a Possible Result of Phagocytosis

1977 ◽  
Vol 14 (4) ◽  
pp. 407-419 ◽  
Author(s):  
E. Gruys ◽  
M. Castaño
Keyword(s):  
Electron Microscopy ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Enzyme Histochemistry ◽  
Amyloid Fibrils ◽  
Light And Electron Microscopy ◽  
Interstitial Cells ◽  
Mesenchymal Cells ◽  
Parallel Arrangement

Vacuoles of mesenchymal cells in the papillae of bovine kidneys with amyloidosis were studied by histochemical electron microscopy for acid phosphatase as a marker for lysosomes. The vacuoles contained parallel amyloid fibrils. The vacuoles of reticular interstitial cells were found to be lysosomes. Vacuoles of macrophage-like cells of the same papillae were positive, partially positive, or negative for the enzyme activity. A suspension of papillary material was injected subcutaneously in rats in a 21-day light and electron microscopy and enzyme histochemistry study. Amyloid was demonstrated in vacuoles of macrophages throughout this period and initially also in neutrophils. In most vacuoles amyloid fibrils were randomly arranged but in others parallel arrangement was demonstrated. Amyloid was only at the inoculation sites. Intralysosomal bovine amyloid may occur in parallel fibrillar arrangement without a definite indication for amyloid production.

Approaches for deciphering the structural basis of low temperature enzyme activity

2000 ◽  
Vol 1543 (2) ◽  
pp. 417-433 ◽  
Author(s):  
Peter P. Sheridan ◽  
Nicholas Panasik ◽  
Jonna M. Coombs ◽  
Jean E. Brenchley
Keyword(s):  
Enzyme Activity ◽  
Low Temperature ◽  
Structural Basis

scholarly journals Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab

1971 ◽  
Vol 121 (3) ◽  
pp. 399-409 ◽  
Author(s):  
Hans W. Behrisch
Keyword(s):  
Skeletal Muscle ◽  
Enzyme Activity ◽  
Low Temperature ◽  
Temperature Sensitive ◽  
King Crab ◽  
Rate Limiting Step ◽  
Low Concentrations ◽  
Higher Temperature ◽  
Paralithodes Camtschatica ◽  
Rate Limiting

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na+ and NH4+ and is then partially inhibited by these cations at concentrations higher than 10mm at 0°, 8° and 15°C. Enzyme activity is stimulated by K+ at 0°C, but is curtailed at 8°C and 15°C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, Km for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature–Km curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in Km for fructose 1,6-diphosphate at 15°C but a 25-fold decrease at 0°C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.

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