Diurnal variation in 5'-nucleotidase activity in rat liver. A quantitative histochemical study

The diurnal variation of 5'-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5'-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5'-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5'-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

The effect of ischemia on glycogen phosphorylase activity in rat liver: a quantitative histochemical study

The effects of ischemia in vitro for 0-60 min at 37 degrees C on glycogen phosphorylase activity in rat liver have been studied under different feeding conditions. Glycogen phosphorylase activity was demonstrated with a recently developed quantitative histochemical method using a semipermeable membrane and the PAS-reaction. The cytophotometrically measured glycogen phosphorylase activity in livers from 24 h-fasted rats was approximately five times the activity in livers from normally fed rats. The activity in periportal areas was about 1.5 times higher than the activity in pericentral areas in livers from starved rats, but more or less evenly distributed in livers from fed rats. Enzyme activity in pericentral areas of livers from 24 h-fasted rats started to decrease after 20 min of ischemia. After 50-60 min of ischemia, the activity was decreased to approximately 25% of the control activity. Livers from normally fed rats showed unchanged activity in periportal and pericentral areas after 10-60 min of ischemia. It has been assumed that the activation of the enzyme was disturbed by ischemia, possibly as a consequence of plasma membrane damage.     

Lead and some other metals in the histochemical demonstration of rat liver glycogen phosphorylase activity

Summary Lead in a concentration of 0.25 mM was tried as a histochemical trapping agent for inorganic phosphate, liberated in the reversible reaction catalyzed by the liver glycogen phosphorylase.The reaction product, formed during the incubation and made visible with ammonium sulphide, was totally extracted with -amylase. Iodine staining after incubation was completely negative.The inhibitory effect on liver phosphorylase activity of several other metals was also studied histochemically. It was found that the inhibition generally increased with the molecular weight and concentration of the metals. It is concluded that Fe⁺⁺ could be useful as a trapping agent instead of lead.     

A quantitative histochemical study of D-amino acid oxidase activity in rat liver in relationship with feeding conditions.

The histochemical method for the demonstration of D-amino acid oxidase activity in rat liver, based on the use of cerium ions and the diaminobenzidine-cobalt-hydrogen peroxide procedure, was improved by the application of unfixed cryostat sections and a semipermeable membrane interposed between section and gelled incubation medium. The amount of final reaction product precipitated in a granular form was about four times higher with this technique in comparison with conventional procedures using fixed sections and aqueous incubation media. The specificity of the reaction was proven by the 70% reduction of the amount of final reaction product when incubating in the presence of substrate and D,L-beta-hydroxybutyrate, a specific inhibitor of D-amino acid oxidase activity. Cytophotometric analysis of liver sections revealed that the specific test minus control reaction was linear with incubation time and section thickness. The Km value of the enzyme of 10.3 +/- 2.7 mM, as determined in periportal areas, is about five times the value found with biochemical methods in liver cell homogenates. The enzyme activity in periportal areas is about five times the activity in pericentral areas. Fasting (24 and 48 hr) induced a significant decrease in D-amino acid activity in periportal and pericentral areas. The possible physiological role of the enzyme in liver is discussed.     

Diurnal variation of HMG-CoA reductase activity in rat liver peroxisomes

The specific activity of hepatic microsomal and peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was determined at different times during a 24 hour cycle from cholestyramine treated rats. The microsomal HMG-CoA reductase activity displayed a peak at D-6 (6th hour of the dark cycle) as previously reported, whereas, the peroxisomal HMG-CoA reductase activity was the highest at L-2 (2nd hour of the light cycle). Immunoblots of the peroxisomal HMG-CoA reductase suggest that the increase in enzyme activity at L-2 is due to changes in enzyme mass. The different cyclic variations observed in microsomal and peroxisomal HMG-CoA reductase activity may suggest different mechanisms of regulation.     

Isolation and study of a liver alpha-amylase. Comparison with glycogen phosphorylase activity

An oligomaltosaccharide-forming amylase has been observed in mice liver crude homogenate. This enzyme has been isolated by binding to amylose. Some of its functional parameters have been studied and compared with those of glycogen phosphorylase demonstrating that amylase activity is not due to a glycogen phosphorylase isoenzyme. It has been further observed that amylase needs Ca2+ of Mg+2 and Cl- for its activity.     

A histochemical study of liver enzymes involved in glycogen metabolism in the X-irradiated rat

Summary The liver enzymes responsible for the breakdown and synthesis of glycogen from glucose have been investigated cytochemically in rats exposed to 1200 rads of x-irradiation. It was found that significant changes occur in their activities and that amylophosphorylase and amylo-1,6-glucosidase (debranching enzyme), both of which are responsible for the conversion of glycogen to glucose, are markedly inhibited by radiation. A significant inhibition of the activity of 1,41,6 transglucosidase (branching enzyme) was also observed. In contrast, the activity of UDPG-glycogen transglucosylase, which is responsible for the in vivo synthesis of 1,4-polysaccharides, was found to be stimulated.     

The nucleotide sequence of rat liver glycogen phosphorylase cDNA.

The nucleotide sequence of a cDNA coding for rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the cDNA are sufficient to encode the total protein as determined by comparison with the liver type of glycogen phosphorylase of man. Human and rat liver glycogen phosphorylase showed 86% homology at the DNA level whereas the deduced amino acid sequence has 93.5% identity.     

Effects of cadmium exposure on glycogen phosphorylase activity in rat placenta as demonstrated by histochemical means

Summary Glycogen phosphorylase (PHO) activity was demonstrated histochemically in unfixed cryostat sections of placentae from cadmium-treated and control rats with the use of the semipermeable membrane technique. Staining of the newly synthesized glycogen was performed by lugol. A high activity was present in glycogen cells, spongiotrophoblast and visceral yolk sac from cadmium-treated and control animals. A low but distinct activity could be demonstrated in placental labyrinth from control rats in late pregnancy. Cadmium-exposed rats showed a considerably higher activity in the labyrinth during this period of pregnancy. The elevated PHO activity and concomitant higher glycogen content indicate a disturbance by exposure to cadmium of placental carbohydrate metabolism from day 18 onwards.     

Effects of cadmium exposure on glycogen phosphorylase activity in rat placenta as demonstrated by histochemical means

Glycogen phosphorylase (PHO) activity was demonstrated histochemically in unfixed cryostat sections of placentae from cadmium-treated and control rats with the use of the semipermeable membrane technique. Staining of the newly synthesized glycogen was performed by lugol. A high activity was present in glycogen cells, spongiotrophoblast and visceral yolk sac from cadmium-treated and control animals. A low but distinct activity could be demonstrated in placental labyrinth from control rats in late pregnancy. Cadmium-exposed rats showed a considerably higher activity in the labyrinth during this period of pregnancy. The elevated PHO activity and concomitant higher glycogen content indicate a disturbance by exposure to cadmium of placental carbohydrate metabolism from day 18 onwards.     

Histochemical demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary A new histochemical method for light microscopic demonstration of liver glycogen phosphorylase activity has been introduced in this study.The method demonstrates phosphorylase activity by precipitating phosphate ions, liberated in the reaction catalyzed by the enzyme, with Fe⁺⁺ present in the incubating medium. The precipitate is visualized as ferrous sulphide.The new glycogen, formed in the same reaction, can also be demonstrated in this method after staining with iodine.The lobular localization of the reaction products obtained according to this method in the liver, corresponds well to that obtained according to earlier methods for the demonstration of only new-formed glycogen.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat.

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Phosphorylation of rat liver glycogen synthase by phosphorylase kinase

Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.     

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

Summary The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.     

Electron microscopic demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary In this study a new electron microscopic method for the demonstration of liver glycogen phosphorylase activity has been presented.Prior to incubation the liver samples were shortly fixed in cold paraformaldehyde. Inorganic phosphate, liberated in the reaction catalyzed by the enzyme, were precipitated with iron (Fe⁺⁺) present in the incubating medium. Postfixation was performed in glutaraldehyde and osmium tetroxide.The ferrous phosphate precipitate was detected electron microscopically in unstained sections.The precipitate was mainly localized to endoplasmic membranes but also in glycogen particles. The method is imperfect in demonstrating phosphorylase activity bound to glycogen particles because of poor preservation of glycogen during treatment.