beta-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor

In order to know if the beta-galactosidase of the rat epididymal fluid, as other secreted acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cellular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact cellular membranes for beta-galactosidase. The results suggest that a phosphorylated carbohydrate of the enzyme is bound by a recognizing site of the cellular membranes different from the phosphomannosyl receptor.     

Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.     

Effect of calcium and magnesium ions on the interaction of corticosterone with the cytosol receptor(s) in the rat brain

The effects of calcium and magnesium ions on the corticosterone binding to rat brain cytosol receptor protein(s) were investigated. The increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, the specific [3H] corticosterone binding increased 1.3-fold and 1.5 respectively. The addition of MnCl2 and KCl did not affect this binding. The binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EDTA and complete inhibition was observed at concentration equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.     

Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors

We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.     

Effects of calcium and magnesium ions on the interaction of corticosterone with rat brain cytosol receptor(s).

The apparent maximum corticosterone binding (B max) with rat brain cytosol and the apparent dissociation constant of this steroid-receptor binding (Kd) estimated with a Scatchard plot was 2.9 X 10(-13) moles/mg cytosol protein and 4.0 X 10(-9) M, respectively. When increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, a specific [3H] corticosterone binding increased 4-fold by CaCl2 at concentrations of 1.0-2.0 mM and 1.5-fold by MgCl2 at concentrations of 0.5-5.0 mM. The addition of MnCl2 and KCl did not affect this binding. Binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EGTA and complete inhibition was observed at concentrations equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.     

L-histidinol dehydrogenase, a Zn2+-metalloenzyme

The enzymatic activity of L-histidinol dehydrogenase from Salmonella typhimurium was stimulated by the inclusion of 0.5 mM MnCl2 in the assay medium. At pH 9.2 the stimulation was correlated with binding of 1 g-atom of 54Mn2+/mol dimer, KD = 37 microM. ZnCl2, which prevented the MnCl2 stimulation, also bound to the enzyme, 1.2 g-atom/mol dimer, KD = 51 microM, and prevented Mn2+ binding. Enzyme activity was lost when histidinol dehydrogenase was incubated in 8 M urea. Reactivation was observed when urea-denatured enzyme was diluted into buffer containing 2-mercaptoethanol but required either MnCl2 or ZnCl2. Histidinol dehydrogenase was inactivated by the transition metal chelator 1,10-phenanthroline or by high levels of 2-mercaptoethanol. The nonchelating 1,7-phenanthroline was not an inactivator, and inactivation by either 1,10-phenanthroline or 2-mercaptoethanol was prevented by MnCl2. Enzyme inactivated by 1,10-phenanthroline could be reactivated by addition of MnCl2 or ZnCl2 in the presence of 2-mercaptoethanol. Reactivation was correlated with the binding of 1.5 g-atom 54Mn2+/mol dimer. Atomic absorption analysis of the native enzyme indicated the presence of 1.65 g-atom Zn/mol dimer, and no Mn was detected. The results demonstrate, therefore, that histidinol dehydrogenase contains two metal binding sites per enzyme dimer, which normally bind Zn2+, but which may bind Mn2+ while retaining enzyme function. Histidinol dehydrogenase is thus the third NAD-linked oxidoreductase in which Zn2+ fulfills an essential structural and/or catalytic role.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization

The structural requirements for oligomerization and the generation of a functional mannose 6-phosphate (Man-6-P) binding site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) were analyzed. Chemical cross-linking studies on affinity-purified CD-MPR and on solubilized membranes containing the receptor indicate that the CD-MPR exists as a homodimer. To determine whether dimer formation is necessary for the generation of a Man-6-P binding site, a cDNA coding for a truncated receptor consisting of only the signal sequence and the extracytoplasmic domain was constructed and expressed in Xenopus laevis oocytes. The expressed protein was completely soluble, monomeric in structure, and capable of binding phosphomannosyl residues. Like the dimeric native receptor, the truncated receptor can release its ligand at low pH. Ligand blot analysis using bovine testes beta-galactosidase showed that the monomeric form of the CD-MPR from bovine liver and testes is capable of binding Man-6-P. These results indicate that the extracytoplasmic domain of the receptor contains all the information necessary for ligand binding as well as for acid-dependent ligand dissociation and that oligomerization is not required for the formation of a functional Man-6-P binding site. Several different mutant CD-MPRs were generated and expressed in X. laevis oocytes to determine what region of the receptor is involved in oligomerization. Chemical cross-linking analyses of these mutant proteins indicate that the transmembrane domain is important for establishing the quaternary structure of the CD-MPR.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

Inhibition and activation of mannan synthesis in Saccharomyces cerevisiae spheroplast lysates.

Mannan synthetase activity in spheroplast lysates prepared from Saccharomyces cerevisiae was measured by following the incorporation of [14C]mannose from guanosine 5'-diphosphate-[14C]mannose into material precipitable with cold 0.3 M perchloric acid. When enzyme activity was assayed at high concentrations of spheroplast lysate protein (10 mg/ml) in the presence of 7.5 mM MnCl2, a severe inhibition was observed. This inhibition could be relieved by preincubation of the spheroplast lysate at 4 degrees C for 16 to 32 h before assay, by repeated freezing and thawing of the spheroplast lysate, or by the omission of MnCl2 from assay mixtures. The addition of ethylenediaminetetraacetic acid or monovalent cations removed inhibition in the presence of Mn2+. No similar inhibition was observed when a washed membrane fraction was substituted for spheroplast lysate as the source of mannan synthetase. The supernatant fluid obtained by centrifuging spheroplast lysate at 100,000 x g, when added to assay mixtures containing either spheroplast lysate preincubated at 4 degrees C or washed membrane fraction, also caused inhibition of enzyme activity. This inhibition required 7.5 mM MnCl2 and was destroyed by heating the supernatant fluid at 60 degrees C for 10 min, or by trypsin treatment at 30 degrees C. These results indicate the existence of a protein inhibitor of mannan synthesis whose inhibitory activity in spheroplast lysates may be modulated by preincubation at low temperature or by varying the available Mn2+ concentration.     

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Renin, a secretory glycoprotein, acquires phosphomannosyl residues

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP- GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine- labeled renin to the Man-6-P receptor as well as direct analysis of [2- 3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.     

Purification and characterization of activated human erythrocyte prolidase

Prolidase (E.C. 3.4.13.9) has been purified 7500-fold to homogeneity from human erythrocytes in a Mn2+-activated form using conventional and fast protein liquid chromatography columns. The procedure includes a 1-h incubation of the crude hemolysate at 50 degrees C with 1 mM MnCl2. Following this novel step, prolidase retains full activity, obviating the requirement for preincubation of each enzyme fraction with Mn2+ prior to assay. Preincubation with MnCl2 does not change the isoelectric point of the enzyme. The molecular weight of the purified enzyme was 58,000 when measured by SDS-PAGE. Western blotting, using rabbit antibody raised to human kidney prolidase, with partially purified erythrocyte enzyme revealed a cross-reacting band at Mr 58,000.     

Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes

To delineate the traffic route through the Golgi apparatus followed by newly synthesized lysosomal enzymes, we subfractionated the Golgi apparatus of rat liver by preparative free-flow electrophoresis into cisternae fractions of increasing content of trans face markers and decreasing contents of markers for the cis face. NADPase was used to mark median cisternae. Beta-Hexosaminidase, the high mannose oligosaccharide processing enzyme, alpha-mannosidase II, the two enzymes involved in the biosynthesis of the phosphomannosyl recognition marker, and the phosphomannosyl receptor itself decreased in specific activity or amount from cis to trans. Additionally, these activities were observed in a fraction consisting predominantly of cisternae, vesicles and tubules derived from trans-most Golgi apparatus elements. These results, along with preliminary pulse-labeling kinetic data for the phosphomannosyl receptor, suggest that lysosomal enzymes enter the Golgi apparatus at the cis face, are phosphorylated, and appear in trans face vesicles by a route whereby the phosphomannosyl receptor bypasses at least some median and/or trans Golgi apparatus cisternae.     

Guanine Nucleotide and Cation Regulation of μ, δ, and k Opioid Receptor Binding: Evidence for Differential Postnatal Development in Rat Brain

A study of the onset of cation and guanine nucleotide regulation of delta, mu, and kappa rat brain opioid receptors during postnatal development was undertaken. Site-specific binding assays were utilized for each receptor type and the effects of 0.5 mM MnCl2, 100 mM NaCl, and/or 50 microM guanosine-5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] were assessed. The most pronounced changes of opioid binding were seen in the presence of Mn2+. In adults, agonist binding to delta sites was stimulated by Mn2+, whereas that to mu sites was not affected and kappa binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on delta sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of kappa site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of delta binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of mu and delta receptor binding by Na+ was greater, and the Mn2+ reversal of this effect was smaller, in the first 2 postnatal weeks than in adults. Gpp(NH)p + Na+ regulation did not change appreciably during the postnatal period. However, Mn2+ reversal of the considerable inhibition elicited by the combination of Na+ and Gpp(HN)p was developmental time-dependent. The data are discussed in terms of multiple sites of interaction for guanine nucleotides and cations.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates.

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100.     

A chemiluminescent assay for quantitation of beta-galactosidase in the femtogram range: application to quantitation of beta-galactosidase in lacZ-transfected cells.

An optimized chemiluminescent assay for beta-galactosidase using a chemiluminescent substrate AMPGD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3,7)]decan]-4- yl)phenyl-beta-D-galactopyranoside) is described. This assay is rapid and sensitive and can detect as little as 2 fg of beta-galactosidase. Its use for the quantitation of beta-galactosidase in cells transfected with lacZ-expressing vectors is described. It is possible to detect a single cell stably expressing lacZ by this technique.