Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

Histochemical method for demonstrating quail mast cell types simultaneously

Abstract

The development and application of selective staining methods for routine detection of mast cells are of considerable interest, because these cells play an important role in health and disease. The composition of cytoplasmic mast cell granules depends on the species and type of mast cell. The study reported here was conducted to investigate the combined use of aldehyde fuchsin (AF) and the Alcian blue-critical electrolyte concentration (AB-CEC) (pH 5.8, 0.3 M MgCl₂) techniques for differentiating avian mast cell subtypes. Tissue samples from skin, intestines, and lungs of six healthy adult quail and two control rats were fixed in Carnoy's solution and 10% formolin for routine histological processing. To determine the staining properties of sulfated glycosaminoglycans (GAGs), a three-step staining technique was applied using berberine sulfate, AF, and AB-CEC. In quail, AF positivity following application of the AB-CEC technique was found only in the lungs, mostly in cells that gave a berberine sulfate-positive reaction, and this positivity was determined to be localized particularly in the nucleus and perinuclear cytoplasm. In other regions, the pale AF staining of cells that did not emit fluorescence when stained with berberine sulfate was determined to be replaced by a blue color after application of AB-CEC. The AF/AB-CEC (pH 5.8, 0.3 M MgCl₂) technique demonstrated that rat and quail mast cells varied in both GAG types and their distribution within the cell. Especially in avian species, this technique can be applied to distinguish mast cells according to their GAG content. It can be used as an alternative to the AB/safranin O staining procedure for differentiating mast cells that contain and lack heparin.     

Iodopolatinate visualization of phospholipids in rat incisor predentine and dentine,compared with malachite green aldehyde

The iodoplatinate (IP) reaction, a selective method for visualization of phospholipids, was applied to the predentine and dentine of rat incisors and compared with malachite green aldehyde (MG) fixation/staining. Spot tests indicated (1) that IP specifically stains phospholipids, but not amino acids, displaying as do phospholipids, quaternary ammonium groups; and (2) phosphatidylserine and sphingomyelin were also stained by MGA. Although this reagent is known to interact with phosphorus, phosphoproteins remained unstained. In the rat incisor, an IP-positive network including granules and thin filaments was seen in predentine in the inter-collagen spaces, in many cases closely associated with collagen fibres and their periodic striations. In dentine, positively stained needle-like structures were located along individual collagen fibres, or at the surface of groups of collagen fibres. This staining pattern was unchanged on sections of material pretreated with acetone, whereas the staining was abolished or markedly reduced when the samples were treated either with chloroform/methanol or phospholipase C prior to the IP reaction. Pretreatment of the samples with hyaluronidase promoted subsequent diffusion of the staining. A very similar staining pattern was observed with MGA, in accordance with earlier reports. The present findings validate the histochemical results reported previously on the distribution and potential role(s) of phospholipids in dentine biomineralization.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Suramin-induced mucopolysaccharidosis in rat incisor

Two weeks after a single injection of suramin, the secretory and post-secretory ameloblasts of the rat incisor were filled with large lysosome-like vacuoles. At the light-microscope level, these vacuoles were positively stained with Alcian blue when MgCl₂ was used at a critical electrolyte concentration varying between 0.1 and 0.3 M, whereas no staining appeared when MgCl₂ varied between 0.7 and 0.9 M. Hyaluronidase digestion markedly reduced but did not totally abolish the staining, indicating that glycosaminoglycans were accumulated inside these vacuoles. Examination of these cells with the electron microscope revealed a polymorphic population of large vesicles, filled to various degrees with cetylpyridinium chloride (CPC)-positive and malachite green aldehyde (MGA)-positive material. The same pattern was observed in secretory odontoblasts but to a lesser extent. In the extracellular matrix, suramin-induced alterations appeared as large defects occurring during enamel formation. In predentin and dentin, the number and/or size of electron-dense aggregates resulting from CPC and MGA fixation, were enhanced in the suramin-injected rats. These aggregates were largely reduced or suppressed respectively by hyaluronidase digestion and chloroform/methanol treatment of the sections. The accumulation of glycosaminoglycans and phospholipids reported here inside ameloblasts and odontoblasts and in predentin and dentin supports the occurrence of suramin-induced mucopolysaccharidosis and lipidosis in this experimental animal model.     

Dentine proteoglycans: composition, ultrastructure and functions

Summary Proteoglycans (PGs) have been visualized in the predentine and dentine with cationic dyes by staining thin sections with Alcian Blue, bismuth nitrate, or using Spicer's high-iron diamine (HID) method. The precise location may be obtained by adding cationic dyes such as Cuprolinic Blue, ruthenium hexammine trichloride or cationic detergent (cetylpyridinium chloride) to the fixative. These methods induced the formation of aggregates which varied in shape and number according to the method used. Rapid freezing followed by freeze-substitution revealed an amorphous ground substance, homogeneously stained with Alcian Blue, located in the predentine between the collagen fibres. These PGs may be involved in transport and diffusion in predentine. In dentine, small granules and needle-like structures were observed along the collagen fibres. This second group of PGs differs in composition, distribution and functions from the predentine PGs. The same distribution was seen when hyaluronidase-gold labelling was used. Labelling with antibodies and autoradiography also gave evidence of two distinct groups of PGs. In predentine, as an hydrated gel, PGs seems to act as mineral inhibitors, whereas immobilized on a surface, as seen at the dentine edge, they act as nucleating agents. The interaction between PGs and phospholipids seems also to play a role in the mineralization process.     

Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Summary Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens containd round granules ( 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl₂ indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC, Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the ³⁵SO₄ labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/ dermatan sulphates comprised about one fifth of the total activity incorporated. The distribution of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.     

The proteoglycan skeleton of the Kurloff body as evidenced by Cuprolinic Blue staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosome organelle, metachromatic towards Toluidine Blue, of a blood cell unique to the guinea pig and called the Kurloff cell. Splenic Kurloff cell from oestrogen-treated guinea pig cells were examined after staining with Cuprolinic Blue, a cationic phthalocyanine-like dye, in the presence of MgCl₂ in a critical electrolyte concentration method. Better results were obtained when the fixation-staining by the glutaraldehyde Cuprinolinic Blue MgCl₂ mixture was preceded by a glutaraldehyde pre-fixation. On light microscopy, Kurloff bodies generally exhibited an overall pink and glassy metachromasia, sometimes with additional darker metachromatic small dots at their peripheries. At the ultrastructural level, the metachromatic central matrix of the Kurloff body usually exhibited, as a major feature, a typical network pattern of ribbon-like or stellate electron-dense precipitates suggesting the presence of a skeleton of Cuprolinic Blue-reactive filamentous structures. Taking into account their high anionicity (as shown by the stability of the dye binding in the presence of 0.3 m MgCl₂) and their susceptibility to chondroitinase ABC, these anionic structures were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell.     

Identification of carrageenan in mammalian tissues: An analytical and histochemical study

Synopsis Methods have been developed for the analytical estimation and histochemical demonstration of carrageeman in the granuloma induced in rats and guinea-pigs by subcutaneous injection of degraded carrageenan.The analytical method for the determination of carrageenan in tissues involved a preliminary clean-up procedure. The tissues were defatted by solvent extraction and incubated with papain and trypsin to remove proteins. Carrageenan and naturally occurring acid mucopolysaccharides were isolated using cetyl pyridinium chloride. The subsequent separation and estimation of carrageenan was carried out by electrophoresis on cellulose acetate paper. Following electrophoresis the cellulose acetate strips were incubated with hyaluronidase to remove acid mucopolysaccharides, and stained with Toluidine Blue. The stained band corresponding to pure degraded carrageenan was quantitated on a scanning densitometer. The method was capable of detecting 0.25 g of degraded carrageenan in tissue.The most suitable method for the histochemical demonstration of carrageenan in paraffin embedded tissues was found to be Alcian Blue at either pH 1 or a CEC (critical electrolyte concentration) of 1.0 M MgCl₂ (pH 5.8). At this pH or CEC, both the carrageenan and the strongly acidic glycosaminoglycans were stained. Prior digestion with hyaluronidase and neuraminidase eliminated the staining of the tissue polysaccharides so that the carrageenan could be visualized within macrophages and in the extracellular space. Mast cell granules retained their staining properties after mucolytic digestion; but morphologically, mast cells could be distinguished from macrophages containing carrageenan.     

Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions

Summary The application of the critical electrolyte concentration (CEC) concept to the differentiation of acidic glycosaminglycans (mucopolysaccharides) is described. Alcian Blue 8GX stains with increasing selectivity as increasing amounts of magnesium chloride are incorporated into the dye solution. Model experiments with pure polyanions, or artifically carboxylated, phosphorylated and sulphated liver sections, showed that binding of dye to carboxylate or phosphate groups ceased at low electrolyte concentrations (< 0.3M) whereas dye continued to be held by sulphate ester groups at concentrations five to ten times as high. The similarity to the well established cetylpyridinium system for polyanion fractionation is discussed.Sections of tissues chosen to contain predominantly or characteristically carboxylated mucins, and/or sulphate ester polyanions showed a staining pattern entirely similar to the model sections. Goblet cell mucin in rat ileum stained at < 0.4M MgCl₂, Cartilage at < 0.6M MgCl₂, mast cells at < 0.75M, and corneal stroma at < 1.0M. These results are in agreement with the known contents of sialo-mucin, chondroitin sulphate, heparin and keratansulphate, respectively. The conditions in which this principle can be used in a practical technique are described.The new and more precise terminology of Jeanloz (1960) is used in preference to the older nomenclature.     

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

Microscopy of the junctional region between human coronal primary and secondary dentine.

The juction between human primary dentine and regular and irregular secondary dentine was examined with a number of different light and electron microscopic techniques. In decalcified material, a narrow band along the innermost surface of the primary dentine stained intensely. The walls of the tubules within the band stained intensely, whereas the tubular walls within the bulk of the primary dentine were not stained. Generally, the walls of the tubules in both types of secondary dentine were also preferentially stained. Although not readily apparent in ground sections, observations of thin sections revealed a dramatic reduction in the number of tubules in regular secondary dentine. Generally, the radiodensity of the intertubular matrix was the same in primary and secondary dentine and the intensely stained band was not seen radiographically. The pulpal ends of the tubules in primary dentine were often occluded with a material having the same radiodensity as peritubular matrix. Both patent and occluded tubules were seen in irregular secondary dentine. Scanning electron microscopy of acid-etched specimens of secondary dentine revealed that some tubules had irregular walls of highly mineralized matrix which was less acid-soluble then the peritubular matrix of primary dentine.     

Separation of β22 dimer from bovine bone collagen

The precise role of the α₂-chain in collagen type I is of considerable scientific interest. Our recent studies demonstrated that the most noticeable difference between type I collagens, which were obtained from bovine hard tissues (bone, dentine) and soft tissues (tendon, skin), was presented in the position of β chain dimers using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The additional band observed both in the bone and dentine collagen was putatively identified as β₂₂ dimer (made of by an intermolecular cross-linking between two α₂-chains). Further investigations carried out on bovine bone and skin collagen, corresponding to hard tissue and soft tissue collagen respectively, confirmed this hypothesis. Successful separation of individual β₂₂ dimer from bone collagen was achieved. The procedure involves molecular-sieve chromatography on a Sephacryl S-400 column followed by differential acetone precipitation. Identification was done by the widely used methods, such as SDS-PAGE and cyanogen bromide (CNBr)-cleaved peptide analysis. It was proposed that the dimer and consequently α₂-chains may play important roles in the morphological and biological differences between hard and soft tissues.     

Formation and Regeneration of Protoplasts of the Actinorhizal Nitrogen-Fixing Actinomycete Frankia

Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 μg/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂. Protoplasts were formed during 15 to 120 min of digestion at 25°C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂ which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl₂, and 5.0 mM CaCl₂. The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1^AG, 27% for strain EAN1pec, and 20% for strain EuI1c.     

Histochemical demonstration of hyaluronic acid molecules by Alcian Blue

Summary Specimens of vitreous humour (monkey eye), Wharton jelly (human umbilical cord) and commercial hyaluronates were immersed in buffered fixative solutions containing either aldehydes and Alcian Blue, or aldehydes and Alcian Blue with MgCl₂ as electrolyte. Two MgCl₂ concentrations were used, 0.025m and 0.3m. Immersion in both solutions induced formation of precipitates which were postfixed in OsO₄, dehydrated and embedded for thin section electron microscopy. The use of the same fixative solution produced morphologically comparable precipitates from all three materials. The precipitates, especially after fixation in the presence of electrolyte, were composed of linear, unbranched filaments, frequently aggregated into bundles. The filaments were considered to be molecules of hyaluronic acid.Part of this work was presented at the 10th Meeting of the European Club for Ophthalmic Fine Structure, Copenhagen, September 3–4, 1982.     

In vitro cartilage formation: effects of 6-diazo-5-oxo-L-norleucine (DON) on glycosaminoglycan and collagen synthesis.

To determine the relationships between glycosaminoglycan (GAG) synthesis and type-specific collagen synthesis, we have investigated mouse limbs cultured in the presence of antiglutamine DON (6-diazo-5-oxo-l-norleucine). When compared to control limbs, ultrastructural examination of the DON-treated limbs shows that newly formed cartilage lacks matrix granules and the collagen fibrils have an altered morphology. Using [³⁵S]sulfate as a precursor, we have found that DON (5 μg/ml) suppresses chondroitin sulfate synthesis to less than 15% of the control level. We have also examined the collagen synthesized in equivalent limbs labeled with [³H]proline. The α-chain patterns from CM-cellulose chromatography were very similar for control and experimental limbs (α1:α2 ～ 7), suggesting that both (α1)₃- and (α1)₂α2-type molecules were being produced. The (α1)₃ molecules in both cases were identified as type II collagen by fractional salt separation and cyanogen bromide peptide mapping on CM-cellulose columns. We conclude that (1) the synthesis of type II collagen can be dissociated from the production of GAG, and (2) environmental influences can be involved in controlling the fibrillogenesis of collagen.     

Glycosaminoglycans in porcine lung: an ultrastructural study using Cupromeronic Blue

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix contributing to the mechanical properties of connective tissues as well as to cell recognition and growth regulation. The ultrastructural localization of GAGs in porcine lung was studied by means of the dye Cupromeronic Blue in the presence of 0.3 M MgCl₂ according to Scott's critical electrolyte concentration technique. GAGs were observed in locations described as follows. Pleura: Dermatan sulphate (DS) and chondroitin sulphate (CS) attached in the region of the d-band of collagen fibrils, interconnecting the fibrils; heparan sulphate (HS) at the surface of elastic fibers and in the basement membrane of the mesothelium and blood vessels. Bronchial cartilage: Abundant amounts of GAGs were observed in three zones: pericellular, in the intercellular matrix and at the perichondrial collagen. By enzyme digestion a superficial cartilage layer with predominantly CS could be distinguished from a deep zone with CS and keratan sulphate. The structure of the large aggregating cartilage proteoglycan was confirmed in situ. Airway epithelium: HS at the whole surface of cilia and microvilli and in the basement membrane of the epithelial cells. Alveolar wall: CS/DS at collagen fibrils, HS at the surface of elastic fibers and in the basement membranes of epithelium and endothelium.     

Cationic dyes reveal proteoglycans on the surface of epithelial and endothelial kidney cells

Summary The glomerular epithelial cells of the rat kidney fixed by vascular perfusion with an aldehyde solution containing either safranine O or alcian blue (and 0.3 M MgCl₂) display filaments which are located close to the outer surface of the plasma membrane. These filaments are similar to those revealed by the same methods in the laminae rarae of the glomerular basement membrane. Alcian blue (and MgCl₂) further demonstrates the presence of anionic sites inside the endothelial cell pores of the glomerular and peritubular capillaries, on the luminal surface of endothelial cells of large renal vessels and along the basolateral surface of the epithelial cells of the Bowman capsule and of the proximal convoluted tubule.Supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 146)     

Histochemical localization of skin glycosaminoglycans during feather development in the chick embryo

Summary The distribution of glycosaminoglycans (GAGs) was studied in embryonic chick skin, using alcian blue staining with critical electrolyte concentration and glycanase treatment, immunofluorescence and transmission electron microscopy. Light microscopy revealed an uneven distribution of sulphated and non-sulphated GAGs at all stages of feather development. Along the dermal-epidermal junction and throughout the depth of the dermis, staining was stronger inside the feathers than in the interplumar skin. With increasing MgCl₂ concentration, the decrease in stain intensity along the dermal-epidermal junction was stronger in interplumar skin than inside feather structures, indicating that sulphated GAGs are more abundant within feathers than in interplumar skin. The same differential sensitivity to electrolyte concentration was noted in the dermis, except at the feather placode stage, when labelling inside the dermal condensation was virtually wiped out at 0.6 M MgCl₂ and higher concentrations, whereas it persisted in the surrounding dermis up to 0.8 M MgCl₂, indicating that the dermal condensation contains a larger amount of hyaluronate than non-feather-forming dermis. Enzyme treatment of sections with Streptomyces hyaluronidase as compared with those treated with chondroitinase ABC corroborated these findings. Immunofluorescent detection of heparan sulphate proteoglycan revealed the presence of the antigen along the dermal-epidermal junction at all stages of feather development, with peaks of brightness in discrete spots of feather structures. Electron microscopy revealed the presence of ruthenium red and tannic acid positive material in the dermal-epidermal junctional zone and inside the dermis. The density of marked granules was somewhat higher in intraplumar than in interplumar regions. These observations demonstrate that certain sulphated and non-sulphated GAGs are distributed in a microheterogeneous manner, which appears to be related to the morphogenetic events of feather development. They are discussed in view of the possible role these components might play in dermal-epidermal interactions. They strengthen the notion, already gained from previous studies on the localization of interstitial collagens and fibronectin, that extracellular matrix components play an important structural and informative role in organogenesis.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.