Polysaccharide synthesis in tissue sections

Summary Biochemical methods for demonstration of enzyme activity are test tube models outside the organization of cell. Their application to the complicated organization of the cell present problems to histochemistry. The morphological and chemical preservation of tissue which is desirable in histochemistry leads to a multiplicity of reactions when test tube methods are applied. For example, the histochemical phosphorylase and glycosyltransferase reactions rest on the assumption that one can distinguish between preexisting glycogen and newly formed polysaccharides. We used frozen dried canine myocardium and liver for examination of the authenticity of histochemical phosphorylase and glycosyltransferase (branching enzyme, UDPG-glycogen transglycosylase) reactions as described in histochemical reference books. We were unable to distinguish between preexisting glycogen and supposedly newly formed polysaccharides with methods presently used for this purpose (Iodine stain, differential digestion with amylases, acid hydrolysis). Tissue without PAS stainable glycogen remained so after substrate incubation. When preexisting glycogen was present, the amounts of stainable polysaccharides after incubation were invariably less. Therefore, we could not prove beyond doubt that any polysaccharide synthesis due to enzyme reaction had oceured. The prescribed controls, perhaps adequate for biochemical test tube reactions, have to be redefined for meaningful histochemical procedures.This work was supported by grant number HE-07605-07 from the National Heart Institute, National Institutes of Health, Bethesda, Md., U.S.A.     

Calcium administration increases calcium-binding protein regucalcin concentration in the liver of rats

The alteration of regucalcin concentrations in the liver and serum of rats administered orally calcium is investigated. Rats received a single oral administration of calcium chloride solution (25, 50 and 75 mg Ca/100 g body weight). The administration of calcium (50 mg/100 g) produced a significant increase in liver regucalcin concentration between 30 and 180 min after the administration, while serum regucalcin concentration was not altered appreciably. The effect of calcium administration increasing liver regucalcin concentration was also seen with the dose of 25 mg/100 g. When liver cytosol prepared from normal rats was incubated for 6 h in the presence of 10 M Ca²⁺, the cytosolic regucalcin concentration at 3 and 6 h of incubation was decreased about 20% (p<0.05) as compared with the value at zero time point, indicating that the presence of Ca²⁺ does not inhibit the decomposition of liver cytosolic regucalcin. Moreover, serum regucalcin concentration was not significantly altered by the incubation for 6 h at 37°C, indicating a stability of regucalcin in rat serum. This suggests that the calcium administration-induced in liver regucalcin concentration is not based on the inhibition of regucalcin release from liver to serum. The present study demonstrates that regucalcin in the liver is clearly increased by calcium administration, presumably due to stimulating the protein synthesis.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

Liver phosphorylase phosphatase

Summary Recent progress in studies on the properties and regulation of liver phosphorylase phosphatase can be divided into four stages. First, isolation from multiple molecular forms of phosphorylase phosphatase, of a single form of catalytic subunit (Mr = 32 000-35 000) which is active toward phosphorylase a and also toward a variety of protein substrates phosphorylated by either cyclic AMP-dependent protein kinase or other protein kinases. This was achieved by rather drastic procedures such as treatment with 80% ethanol at room temperature, incubation with 6 M urea, freeze-thawing in the presence of 0.2 M mercaptoethanol, or digestion by trypsin. These treatments caused concomitantly large enhancement of phosphorylase phosphatase activity, and the hypothesis was proposed that an inactive form of phosphorylase phosphatase existed as complexes of a catalytic subunit and inhibitory proteins. Second, it was discovered that liver and muscle extracts contain trypsin-labile proteins which, after heating at 90 °C, inhibited the catalytic subunit of phosphorylase phosphatase. Two types of protein inhibitors were identified: inhibitor-I was phosphorylated and activated by cyclic AMP-dependent protein kinase, whereas inhibitor-2 was not phosphorylated. Although inhibitor-1 has been implicated in hormonal regulation of glycogen metabolism in skeletal muscle, a similar role of protein inhibitors in the regulation of phosphorylase phosphatase in the liver has not been demonstrated and the physiological role of the inhibitor is questionable.Third, it has been demonstrated that liver phosphorylase phosphatase is reversibly inactivated and regulated by glutathione disulfide (GSSG) at the catalytic subunit level. Liver phosphorylase phosphatase contains, per mole of catalytic subunit, two sulfhydryl groups, one of which reacts with GSSG to form mixed disulfide with consequent inactivation of the enzyme. The inactivated enzyme can be reactivated by glutathione(GSH) or other sulfhydryl compounds through a reverse reaction. Injection of GSSG into the portal vein of rabbits caused a rapid increase in phosphorylase-a activity in the liver, suggesting that GSSG is involved in regulation of phosphorylase activity in vivo.Finally, current evidence suggests that liver phosphorylase phosphatase exists in the native state in a high molecular weight form which consists of the catalytic subunit and other regulatory subunits. One such enzyme species could be a 260 000-dalton protein composed of three different types of subunit, termed , and , or a 160 000-dalton protein composed of and subunits. The a subunit (Mr = 35 000) appears to be identical to the multifunctional catalytic subunit, whereas the (Mr = 69 000) and (Mr = 58 000) subunits are catalytically inactive but can modify the catalytic a subunit. It seems likely that the substrate specificity and catalytic activity of the subunit is considerably altered when it is part of larger complexes with other regulatory subunits ( and ). It has also been suggested that in addition to the native form of phosphorylase phosphatase, liver contains a considerably large amount of latent phosphorylase phosphatase, the catalytic activity of which could be revealed only by treatment with trypsin or ethanol.     

Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2+-independent, autophosphorylation-dependent protein kinase

In a previous report (Yu and Yang,Biochem. Biophys. Res. Commun. 207, 140–147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca²⁺-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver (Yanget al, J. Biol. Chem. 262, 7034–7040, 9421–9427 (1987)]. In this report, the effect of Mg²⁺ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg²⁺ ion used. Phosphorylation of phosphorylase b kinase at high Mg²⁺ ion (>9 mM) is 2–3 times higher than that observed at low Mg²⁺ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg²⁺ ion. Analysis of the phosphorylation stoichiometry of and subunits of phosphorylase b kinase at different Mg²⁺ ion concentrations further reveals that the phosphorylation level of the subunit remains almost unchanged, whereas the phosphorylation level of the subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg²⁺ ion. Taken together, the results provide initial evidence that Mg²⁺ ion may specifically render thea subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.     

Regulation of glycogen phosphorylase in fat body ofCecropia silkmoth pupae

Summary Glycogen phosphorylase of pupal fat body of the silkmoth,Hyalophora cecropia, and its activation by different stimuli have been studied. Spectrophotometric assay in the direction of glycogenolysis, used in most of the experiments, indicated higher amounts of phosphorylasea than assay by release of P_i from glucose-1-phosphate; both assays, however, estimated changes in proportion of phosphorylasea equally. TheK _ms for P_i were estimated as 5 mM for phosphorylasea in the absence of AMP and 18 mM for phosphorylaseb with 2 mM AMP.When diapausing pupae were held at 4°C, fat body phosphorylase was quickly activated by conversion to thea form up to about 50% of the total, and then declined again after 30 days, when glycerol had accumulated in the hemolymph. Cold activation in vivo was quickly reversed at 25°C. Removal of the brain did not prevent cold activation. After storage at 15°C, sensitivity to cold activation was diminished. Locusts and crickets also showed activation of phosphorylase after chilling.Exposure of fat body to air, transfer to Ringer solution, or physical agitation, caused activation of phosphorylase which is classed as shock activation. After about 1 h incubation in Ringer at 25°C, this effect reversed spontaneously. Activation also occurred in fat body in vitro after transfer to 0°C (cold activation), and was reversed at 25°C. The previously reported inhibition of activation by glycerol, however, could not be consistently reproduced.In fat body homogenates, phosphorylaseb was converted to phosphorylasea by incubation with ATP and Mg²⁺, which indicates activity of phosphorylase kinase. In preparations treated with Sephadex G-25 and then incubated, the reverse conversion took place, which was inhibited by fluoride, and indicates activity of phosphorylase phosphatase.Cyclic AMP added to fat body in vitro, or theophylline either in vivo or in vitro, stimulated the activation of phosphorylase. In fat body in vitro, shock activation was paralleled by elevation of tissue cyclic AMP, whereas cold activation was not. Cyclic GMP did not stimulate activation, and showed no significant changes in tissue levels.It is concluded that the conversion of silkmoth pupal fat body phosphorylaseb to phosphorylasea can be stimulated by a shock-initiated mechanism involving cyclic AMP and a distinct cold-initiated mechanism independent of cyclic AMP.Abbreviations DTT dithiothreitol - cyclic AMP 3,5-cyclic adenosine monophosphate - cyclic GMP 3,5-cyclic guanosine monophosphate - P _i inorganic phosphate This investigation was begun in the Department of Biology, Yale University, New Haven, Connecticut, USA     

Isolation and characterisation of an acid phosphatase interfering with phosphorylase determinations in crude extracts from yeast

In phosphorylase assays in crude yeast extracts with glucose-1-phosphate (G-1-P) as substrate, 25–30% of the P_i-liberating activity could not be inhibited by antibodies against yeast phosphorylase and were attributed to the action of phosphatases. During phosphorylase preparation from baker's yeast (Saccharomyces cerevisiae), a phosphatase, molecular weight 45000±5000, with high specificity for G-1-P, pH-optimum 5.6, was isolated which appeared to be responsible for the interference. It did not hydrolyze other glycolytic intermediates, pyrophosphate or adenylates. No activation by Mg²⁺ or inhibition by (+)-tartrate, and only 40% inhibition by 50 mM F^- were observed, 5,5 dithiobis-(nitrobenzoic acid) (10mM) inactivated the enzyme completely. Its affinity for G-1-P was very low (K _m=40 mM). Consequently, it mainly interfered with the phosphorylase assay in the amylose synthesizing reaction, in which high G-1-P-concentrations have to be used. For phosphorylase assays in crude extracts, measurement of the phosphorolytic activity is recommended, in which the concentration of G-1-P is kept sufficiently low.Abbreviations G-1-P Glucose-1-phosphate - (NbS)₂ 5,5 dithiobis-(2-nitrobenzoic acid) - SDS Sodium dodecylsulfate     

Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

Interaction of hydrolytic and phosphorolytic enzymes of starch metabolism in Kalanchoë daigremontiana

The degradation of starch by a protein fraction of Kalanchoë daigremontiana Hamet et Perrier, obtained by ammoniumsulfate precipitation (30–70%), was found to be catalyzed by -and -amylase (EC 3.2.1.1 and EC 3.2.1.2, respectively) and by starch phosphorylase (EC 2.4.1.1). The activity of these enzymes was determined by chromatographic analysis of the reaction products; separation and identification of -amylase was accomplished by heat-inactivation of -amylase and -glucosidase. When the interaction of amylolytic and phosphorolytic enzymes was comparatively studied, it was found that without inorganic phosphorus in the reaction mixture, ¹⁴C-starch was converted predominantly to maltose and glucose; supplementation with 1–10 mM orthophosphate (Pi) resulted in an increase in glucose-1-phosphate formation and a concomitant reduction of maltose production. Since the total volume of starch degradation remained approximately constant, Pi apparently inhibits -amylase (Ki about 3 mM Pi). Thus, free Pi in the cell participates in the regulation of starch catabolism, serving as a substrate for starch phosphorylase while simultaneously reducing the production of maltose. With respect to glucan synthesis, adenosinediphosphoglucose--1,4-glucosyltransferase (EC 2.4.1.22), maltose phosphorylase and maltoseglucosyltransferase were also found to be active. The last-named enzyme catalyzes an exchange between dextrins and is considered to provide primer carbohydrates for the synthesis of polyglucans.Abbreviations ADPG adenosinediphosphoglucose - G1P glucose-1-phosphate - PEG polyethylenglycol - PEP phosphoenolpyruvate - Pi orthophosphate     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.     

Effects of ischaemia and reperfusion on NADH coenzyme Q reductase activity in rat liver

NADH coenzyme Q reductase (EC 1.6.5.3) has been suggested in the literature to be inactivated by ischaemia. In the present study, NADH coenzyme Q reductase activity was localized in unfixed cryostat sections of ischaemic rat livers and quantified using image analysis. In vitro ischaemia was induced by storage of rat liver fragments for 30, 60, and 120min at 37°C. In vivo ischaemia was provoked by clamping the afferent vessels of median and left lateral liver lobes for 60min followed by 30, 60 and 180min of reperfusion. NADH coenzyme Q reductase activity was demonstrated with the tetrazolium salt method in the presence of polyvinyl alcohol. Final reaction product was found in liver parenchymal cells and its distribution was homogeneous within liver lobules. Only low amounts of final reaction product were formed when the incubation was performed in the absence of the substrate NADH. A non-linear relation was found between the absorbance and incubation time when the reaction was performed in the presence of NADH. Therefore, the initial velocity was taken as the true rate of enzyme activity. A linear relationship was found for the initial velocity and section thickness up to 6µm followed by a levelling off. Electron microscopically, NADH coenzyme Q reductase activity was localized at the outer and inner membranes of mitochondria. In vitro ischaemia up to 120min did not affect NADH coenzyme Q reductase activity. At 30min reperfusion after in vivo ischaemia for 60min enzyme activity was slightly decreased in certain foci which also showed diminished lactate dehydrogenase activity. A further decrease of enzyme activities in foci was observed at 180min reperfusion after ischaemia. It is concluded that NADH coenzyme Q reductase activity is not sensitive to ischaemia. Furthermore, it is likely that the enzyme leaks from liver parenchymal cells into the circulation during reperfusion after ischaemia.     

In vitro electrochemical detection of wheat allergen using rat basophilic leukaemia (RBL-1) cells

We have investigated an electrochemical method of detecting foods that cause an allergic reaction. Rat basophilic leukaemia (RBL-1) cells were sensitized with serum from a rat that was allergic to wheat. A sample containing the protein fraction of a food was added to the cells and incubated. The cells were immobilized on a membrane filter and attached to a basalplane pyrolytic graphite electrode. When a potential was applied in the range 0–1.0 V relative to a saturated calomel electrode, an anodic peak current appeared at around 0.33 V. This peak current, attributed to serotonin, increased with time, and the maximum current (0.5 A) was obtained 20–25 min of incubation. The response of the RBL-1 cells was specific to the protein fraction of wheat. The peak current increased linearly with increasing protein concentration in the range of 0.01–0.5 g ml^–1. These results suggest that the concentration of the protein bringing about the allergic reaction can be determined by cyclic voltammetry within 25 min. This method is more sensitive than the conventional skin tests.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

Iodide,thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity

Summary The necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation in vitro and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm.It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation in vitro is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections.The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN^– or CNS^–. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN^–, should be substituted for I^– in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.This work was supported by the grant of Research association of Slovenia and by the NIH grant PL 480 N° 02-008-N     

A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver

Summary The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light-and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mm diaminobenzidine, 44 mm hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 m. Catalase in rat liver showed a K_m value of 2.0 mm for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mm. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.