Biochemical and cytochemical localization of ATPases on the membranes of the electrocyte of Electrophorus electricus

Summary The localization of (Na⁺-K⁺) ATPase in the intact electrocyte of the electric organ of Electrophorus electricus (L.) and its subcellular fractions was investigated by biochemical and cytochemical methods. The distribution of AChE activity in the subcellular fractions was also comparatively analysed with this enzyme serving as a marker of the innervated membranes of the electrocyte. After application of cytochemical method of Farquhar and Palade to glutaraldehyde-fixed tissue, reaction was observed only at the membranes of vesicles localized at the periphery of the electrocyte. Previously fixed electrocytes, incubated in Ernst's medium showed reaction only at the vesicles whereas in unfixed tissue reaction also appeared at other membranes (surface and invaginations) of the anterior and posterior faces. This reaction was significantly inhibited in the presence of ouabain or in the absence of K⁺. Inhibition of Na⁺-K⁺-ATPase by glutaraldehyde fixation was also confirmed by biochemical analysis.This investigation has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Conselho de Ensino e Pesquisa da UFRJ and FINEP (FNDCT-375/CT)     

Electron cytochemical study of the muscle cell surface

Summary The lectins wheat germ agglutinin and limulus polyphemus were used as cytochemical probes to study the ultrastructural localization of sialic acid at the cell surface of rat muscle fibers. In addition cytochemical studies employing strontium as an electron-dense marker were also carried out to investigate cation binding sites at the muscle cell surface. The results showed binding of the lectins to the glycocalyx, caveolae and the basal lamina of the muscle fibers. These binding sites matched the ones observed in the cytochemical studies using strontium as a marker. Based on these observations we suggest that the glycocalyx, caveolae and the basal lamina of the muscle fiber may be involved in the binding of Ca⁺⁺ and that significant amounts of Ca⁺⁺ may be normally present at the muscle cell surface.Supported by a grant from the Muscular Dystrophy Association and by Center Grant NS-1176 from the National Institute of Neurological and Communicative Disorders and Stroke     

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague--Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 μmol^?1 min^?1 100 g^?1 body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrink age were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5′-nucleotidase, and extracellular adenosine production, employing 141Ce3+ as a capturing agent

Summary A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing¹⁴¹Ce³⁺ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5′-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce³⁺ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.     

Cytochemical localization of K+-dependent p-nitrophenyl phosphatase and adenylate cyclase by using one-step method in human washed platelets

Summary Ultrastructural cytochemical localization of ouabain-sensitive, potassium dependent p-nitrophenyl phosphatase (K⁺-NPPase) of the Na⁺-/K⁺-ATPase complex and adenylate cyclase (cAMPase) activities, in washed inactivated human platelets, are described. The one-step lead-citrate method, under similar incubation conditions, was used to determine both activities. K⁺-NPPase appeared in both plasma membrane and the surface-connected canalicular system (SCCS) of the platelets. These data suggest a uniform distribution of the enzyme throughout membrane systems which are in contact with the external medium. cAMPase activity was strictly localized in tubules of the dense tubular system (DTS) when incubation medium contained prostaglandin E₁, prostaglandin D₂ or forskolin, at concentrations known to stimulate the enzyme in platelets that are intact. This fact and the inhibition of cytochemical reaction by thrombin confirm that the one-step lead-citrate method is a useful procedure in determining adenylate cyclase, abolishing the unfavorable conditions of previously reported methods.     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague--Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 μmol⁻¹ min⁻¹ 100 g⁻¹ body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrink age were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features This revised version was published online in November 2006 with corrections to the Cover Date.     

Cellular localization of polyamines: Cytochemical and ultrastructural methods providing new clues to polyamine function in ram spermatozoa

Summary— Polyamine binding sites have been localized in ram spermatozoa using biochemical and cytochemical tools. Incubating the cells with ¹⁴C-spermine and determining its distribution after sonication and differential centrifugation, revealed that 60% of the radioactive spermine was localized in the head, 21.5% in the tail and about 9% in the plasma membrane. A polyamine specific cytochemical staining by the formaldehyde-fluorescamine method, revealed that most of the polyamines were localized in the midpiece, where the cell mitochondria are located, and in the acrosome region. Two additional studies used electron microscopy, employing polycationic colloidal gold and spermine-ferritin as cytochemical markers. The most sensitive and specific method was the staining of the cells with ferritin-spermine whose synthesis is described in this study. The outer acrosomal membrane was the preferential site for spermine binding which was densely distributed in a highly orderly pattern. There was a sparse distribution of spermine binding sites on the plasma membrane surrounding the acrosome and none on the post acrosomal region. The role of spermine in the acrosome reaction and Ca²⁺ fluxes in sperm cells is discussed.     

Assessment of a strontium capture technique for cytochemical localization of potassium-dependent phosphatase in Malpighian tubules of Locusta

Abstract A strontium capture method, using p-nitrophenyl phosphate as substrate, was used to determine the subcellular localization of (Na⁺ + K⁺)-ATPase activity in Malpighian tubules of Locusta migratoria L. Ultrastructural studies revealed that (Na⁺ + K⁺)-ATPase activity was restricted to the basolateral plasma membranes with little evidence of activity associated with the apical microvilli. In contrast, alkaline phosphatase activity was specifically associated with the apical cell membrane. Biochemical assays of fixed and non-fixed tubule homogenates were used to evaluate the p-nitrophenyl phosphate-strontium procedure for localization of the phosphatase component of (Na⁺ + K⁺)-ATPase. No significant potassium-dependent, ouabain-sensitive p-nitrophenyl phosphatase activity was demonstrated in homogenates under conditions necessary for the cytochemical procedure, viz fixation, pH 9.0 and the presence of strontium. The significance of the biochemical results are discussed in relation to the validity of such cytochemical techniques for (Na⁺ + K⁺)-ATPase localization.     

Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry

Summary A cytochemical study of gastric K⁺-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K⁺-stimulated phosphoprotein phosphatase of H-K-ATPase system, has been made by a new cytochemical method.Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p-nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K-ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method.These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane.In honour of Prof. P. van DuijnPart of this paper was presented at the 24th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Nagoya, October 27–28, 1983 (Ogawa KS, Fujimoto K, Ogawa K (1983) A new lead citrate method for the cytochemical demonstration of the H⁺–K⁺-ATPase with p-NPP as a substrate. Acta Histochem Cytochem 16:662)This study was supported by Grants-in-Aid for Encouragement of Young Scientists No. 60770019 to K. Fujimoto from the Ministry of Education, Science and Culture, the Japanese Government     

Identification of Spermatozoa in Criminological Investigations

Materials used for study were viral smears or ultra-thin sections containing viral cell inclusions. They were stained with the Feulgen reaction and other cytochemical procedures. Stained preparations were dried and then shadow-cast with metallic chromium for 30 seconds in a bell jar with a vacuum of at least 0.1 µ (10^?4mm.) of mercury, and placed at a shadowing angle of 10–12°. Shadow-cast preparations were cleared with xylene and mounted in Canada balsam. Dried smears or deparaffinized sections without staining were suited to this method also. A virus which stained indistinctly with cytochemical procedures alone could be adapted to visible light microscopy by shadowing, and in addition, used for observations on its chemical composition.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

CYTOCHEMICAL LOCALIZATION OF 5''-NUCLEOTIDASE IN SUBCELLULAR FRACTIONS ISOLATED FROM RAT LIVER : I. The Origin of 5''-Nucleotidase Activity in Microsomes

A procedure has been developed for the cytochemical localization of 5'-nucleotidase in isolated, unfixed, rat liver microsomes. Membranes were incubated with adenosine 5'-phosphate (5'-AMP) and Pb(NO₃)₂ and then isolated on sucrose density gradients: all the phosphate released was recovered with the membranes by this procedure. Adenosine 2'-phosphate (2'-AMP) and adenosine 3', 5'-cyclic phosphate (3',5'-AMP) were shown to be competitive inhibitors, but not substrates, for purified 5'-nucleotidase and were employed to determine the specificity of the cytochemical reaction. It was found that the incubation conditions for the cytochemical assay did not affect the specificity of 5'-nucleotidase. Microsomes incubated as controls with Pb²⁺, or Pb²⁺ and 2'-AMP or 3',5'-AMP were of the same density, although slightly denser than microsomes incubated without Pb²⁺, and were unassociated with lead precipitate when examined by electron microscopy; microsomes incubated with Pb²⁺ and 5'-AMP were much denser and were stained heterogeneously with lead phosphate when examined by electron microscopy. Precipitates formed artificially from Pb²⁺ and inorganic phosphate did not resemble the reaction product. Microsomes were, therefore, separated on sucrose gradients and the subfractions were examined cytochemically. Lead precipitates were associated with the majority of rough-surfaced vesicles, and the reaction product was distributed heterogeneously in all fractions. Vesicles which stained like the membranes of the bile canaliculi in isolated plasma membranes were observed in the lightest subfraction. The reaction product was localized on the outside surface of the microsomal membranes, and was solubilized by low concentrations of ethylenediaminetetraacetic acid. It is concluded that 5'-nucleotidase is present in the endoplasmic reticulum and that the microsome fraction contains, in addition, vesicles derived from the plasma membrane.     

Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid worm,Phascolosoma scolops

Summary Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K⁺-contracture was dependent on external Ca²⁺ concentration and the contracture disappeared in Ca²⁺-free solution. The K⁺-contracture was suppressed by application of procaine and Mn²⁺. Caffeine induced contracture even when external Ca²⁺ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO₄ solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca²⁺ as well as by the release of Ca²⁺ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.     

Esterase activity of Aspergillus niger in continuous culture

In citrate limiting medium the esterase activity of Aspergillus niger had a maximum value at the lowest dilution rate (D=0.013 h^-1) and at all higher dilution rates progressively decreased in activity. In glucose limiting medium the esterase activity values were always lower than in citrate limiting medium and did not show much variation with varying dilution rate. Electrophoresis of cell free extracts from all dilution rates revealed a multimolecular esterase profile only at D=0.013 h^-1 in citrate limiting medium, which was also the only dilution rate to support good conidiation. The increase in esterase activity at D=0.013 h^-1 was observed cytochemically to occur in the phialides. No cytochemical esterase staining occurred in the vegetative cultures at all other dilution rates.     

Calcium stimulation of glucose-6-phosphate dehydrogenase activity in shoot apices of Spinacia oleracea during floral evocation

The earliest biochemical marker of floral evocation in the shoot apex of S. oleracea is the doubling of the rate of glucose-6-phosphate dehydrogenase (G6PD) activity 12–15 h after transfer of 4-week-old plants from short days to continuous light i.e. 1–2 h after the leaves are raised to the floral state. Quantitative cytochemical analysis of G6PD activity in the vegetative apices showed that addition of 10⁻⁷ M Ca²⁺ to the cytochemical enzyme reaction medium for G6PD activity raises the rate of enzyme activity to that seen in the induced apices. Higher concentrations of Ca²⁺ result in G6PD inhibition in the vegetative apices and any added Ca²⁺ at concentrations of 10⁻⁷ M or higher inhibit the G6PD activity seen in both the induced apices and leaf primordia of both types of apex. The addition of EGTA abolishes the cytochemical reaction. The ability of the Ca²⁺ to activate the G6PD activity in addition to the incubation medium occurs during the periods of 8–11 h of continuous light, but is already lost by 12 h when no change is achieved by Ca²⁺ treatment. This can be interpreted as indicating a point in time close to the moment of floral evocation. A model is proposed in which Ca²⁺ is able to activate the inactivated-G6PD molecules in the vegetative apex through increased Ca²⁺ flux possibly through the action of plasmalemmal Ca²⁺-ATPase activity as part of the floral evocation process. © 1998 John Wiley & Sons, Ltd.     

The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view

The NADPH oxidase complex catalyzes the formation of superoxide (O₂ ^–) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed.Portions of this review were presented at the 36th Symposium of the Society for Histochemistry, 21 September 1994, Heidelberg, Germany     

Cytochemical localization of ouabain-sensitive,K+-dependent p-nitrophenylphosphatase and Ca++-stimulated adenosine triphosphatase activities in human parotid and submandibular glands

Summary K⁺-dependent p-nitrophenylphosphatase (pNPPase) and Ca⁺⁺-stimulated adenosine triphosphatase (ATPase) activities were studied in human parotid and submandibular glands using cytochemical methods at the ultrastructural level. In both glands, only the striated-duct epithelium showed K⁺-pNPPase reaction product, thereby indicating the localization of Na⁺, K⁺-ATPase. The precipitate was concentrated on the deep invaginations of the basolateral plasma membranes, in close association with their cytoplasmic surface. Ca⁺⁺-ATPase activity was also found on the basolateral plasma membranes, but two striking differences from the K⁺-pNPPase distribution were observed: firstly, Ca⁺⁺-ATPase appeared in both acinar and ductal cells, and secondly, it was localized on the outer side of the plasma membranes.     

Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells

Summary A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37° C. The incubation medium contained HRP, lysozyme and Ca²⁺ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37° C in HEPES buffer containing 20 mM Ca²⁺. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37° C in the absence of Ca²⁺, and was measured by spectrophotometry. The addition of 20 mM Ca²⁺ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca²⁺. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations. When dried HeLa cells, fixed in methanol, were incubated with HRP, lysozyme and Ca²⁺, without being subsequently incubated with Ca²⁺-containing buffer solution, HRP was also bound to membranes of intracellular granules. Cytochemical observations showed that UDP-galactose and chitotriose competed with the binding of HRP to most of these intracellular membranes whereas CMP-neuraminic acid and gangliosides did not. The possible binding of HRP to galactosyltransferase or sialyltransferase on cellular membranes is discussed.