Association of inorganic pyrophosphatase activity with normal calcification of rat costal cartilage in vivo

1. Dialysed extracts of rat costal cartilage were shown to possess an enzyme that hydrolyses inorganic pyrophosphate. 2. Inorganic pyrophosphatase activity assayed in the presence of 2mm substrate was maximal at pH6.8. 3. Mg(2+) was essential for activity, which was greatest with 10mm or higher concentrations of Mg(2+). 4. Extracts prepared from cartilage taken from suckling rats (<20g.) showed little or no hydrolytic activity, but as rat weight increased inorganic pyrophosphatase activity was detected, increased to a maximum in tissue from animals weighing about 40g., and then rapidly declined. 5. The increase in inorganic pyrophosphatase activity was associated with an increase in the uptake of (45)Ca by the cartilage in vivo. 6. Accumulation of calcium, inorganic phosphate and magnesium occurred when inorganic pyrophosphatase activity was at its maximum. 7. Alkaline phosphatase activity, measured in the same extracts used to determine pyrophosphatase activity, was highest in the tissues of the animals weighing <20g., and decreased as inorganic pyrophosphatase activity increased to its maximum. 8. There was no direct relationship between alkaline phosphatase activity and the onset of calcification.     

Resolution, purification and characterization of the orthophosphate releasing activities from fracture callus calcifying cartilage.

Callus calcifying cartilage alkaline phosphatase was resolved by DEAE-cellulose column chromatography into two distinct phsophatase activities. The phosphatase activity which was eluted first from the column, (phosphatase I), was active towards a variety of phosphate esters, sodium pyrophosphatase and several linear polyphosphates, while the second phosphatase activity , (phosphatase II), was active toward simple phosphate esters but not towards sodium pyrophosphate and linear oligo or polyphosphates. All the phosphate esters, sodium pyrophosphate and polyphosphates at higher concentrations were inhibitory for phosphatase I. The modulating effects of magnesium, calcium, zinc and other phosphatase modulators have been investigated. Both phosphatases from callus calcifying cartilage were found to be substrates of neuraminidase with sialic acid as the product. Besides the difference in their specificity, the phosphatases were found to be immunologically different and to have different molecular weights, strong indication that they are different enzymes.     

Phosphohydrolases of the phytopathogenic fungus Phytophthora infestans (Mont) de Bary]

ATPase, pyrophosphatase and tripolyhosphatase activities were found in a cell-free Phytophtora infestans micelium extract. No polyphosphatase activity, hydrolyzing high molecular weigh polyphosphates to orthophosphate, was observed in the fungi. It was demonstrated that, unlike ATPase, the activity of pyrophosphatase was inhibited by Ca2+ at concentrations from 0.1 to 20 mM, and it was considerably decreased in the presence of a Ca2+ transport inhibitor, ruthenium red (0.01--0.1 mM). Possible relation of Ph. infestans pyrophosphatase activity with the process of active calcium transport is suggested.     

Regulation of in vitro calcium phosphate mineralization by combinatorially selected hydroxyapatite-binding peptides

We report selection and characterization of hydroxyapatite-binding heptapeptides from a peptide-phage library and demonstrate the effects of two peptides, with different binding affinities and structural properties, on the mineralization of calcium phosphate mineral. In vitro mineralization studies carried out using one strong- and one weak-binding peptide, HABP1 and HABP2, respectively, revealed that the former exhibited a drastic outcome on mineralization kinetics and particle morphology. Strong-binding peptide yielded significantly larger crystals, as observed by electron microscopy, in comparison to those formed in the presence of a weak-binding peptide or in the negative control. Molecular structural studies carried out by circular dichroism revealed that HABP1 and HABP2 differed in their secondary structure and conformational stability. The results indicate that sequence, structure, and molecular stability strongly influence the mineralization activity of these peptides. The implication of the research is that the combinatorially selected short-sequence peptides may be used in the restoration or regeneration of hard tissues through their control over of the formation of calcium phosphate biominerals.     

Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes

Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.     

Role of matrix vesicles in calcification.

The role of matrix vesicles in the calcification process was investigated in vitro. Isolated vesicles were unable to transport calcium actively. The ATPase activity was not stimulated by calcium in the presence of an optimal magnesium concentration. At a physiological substrate concentration of pyrophosphate, the pyrophosphatase had a pH optimum around 7.0. The vesicles nucleated calcium phosphate precipitation independently of the presence of hydrolyzable phosphate compounds. It is suggested that vesicles induce calcification by nucleating calcium phosphate precipitation and through the local destruction of pyrophosphate, a crystallization inhibitor.     

Alterations in the sensing and transport of phosphate and calcium by differentiating chondrocytes

During endochondral bone formation and fracture healing, cells committed to chondrogenesis undergo a temporally restricted program of differentiation that is characterized by sequential changes in their phenotype and gene expression. This results in the manufacture, remodeling, and mineralization of a cartilage template on which bone is laid down. Articular chondrocytes undergo a similar but restricted differentiation program that does not proceed to mineralization, except in pathologic conditions such as osteoarthritis. The pathogenesis of disorders of cartilage development and metabolism, including osteochondrodysplasia, fracture non-union, and osteoarthritis remain poorly defined. We used the CFK2 model to examine the potential roles of phosphate and calcium ions in the regulatory pathways that mediate chondrogenesis and cartilage maturation. Differentiation was monitored over a 4-week period using a combination of morphological, biochemical, and molecular markers that have been characterized in vivo and in vitro. CFK2 cells expressed the type III sodium-dependent phosphate transporters Glvr-1 and Ram-1, as well as a calcium-sensing mechanism. Regulated expression and activity of Glvr-1 by extracellular phosphate and parathyroid hormone-related protein was restricted to an early stage of CFK2 differentiation, as evidenced by expression of type II collagen, proteoglycan, and Ihh. On the other hand, regulated expression and activity of a calcium-sensing receptor by extracellular calcium was most evident after 2 weeks of differentiation, concomitant with an increase in type X collagen expression, alkaline phosphatase activity and parathyroid hormone/parathyroid hormone-related protein receptor expression. On the basis of these temporally restricted changes in the sensing and transport of phosphate and calcium, we predict that extracellular phosphate plays a role in the commitment of chondrogenic cells to differentiation, whereas extracellular calcium plays a role at a later stage in their differentiation program.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO4: Ligand-induced molecular asymmetry

The three-dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Patterson's search technique and refined to an R-factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α₃)₂ into α₃′α₃″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re-arrangements for pyrophosphatase function is discussed.     

Mineralisation of soft and hard tissues and the stability of biofluids

Evidence is provided from studies on natural and artificial biofluids that the sequestration of amorphous calcium phosphate by peptides or proteins to form nanocluster complexes is of general importance in the control of physiological calcification. A naturally occurring mixture of osteopontin peptides was shown, by light and neutron scattering, to form calcium phosphate nanoclusters with a core–shell structure. In blood serum and stimulated saliva, an invariant calcium phosphate ion activity product was found which corresponds closely in form and magnitude to the ion activity product observed in solutions of these osteopontin nanoclusters. This suggests that types of nanocluster complexes are present in these biofluids as well as in milk. Precipitation of amorphous calcium phosphate from artificial blood serum, urine and saliva was determined as a function of pH and the concentration of osteopontin or casein phosphopeptides. The position of the boundary between stability and precipitation was found to agree quantitatively with the theory of nanocluster formation. Artificial biofluids were prepared that closely matched their natural counterparts in calcium and phosphate concentrations, pH, saturation, ionic strength and osmolality. Such fluids, stabilised by a low concentration of sequestering phosphopeptides, were found to be highly stable and may have a number of beneficial applications in medicine.     

Distribution and Immunologic Cross Reactivity of a Phosphohydrolytic Activity of Calcifying Cartilage and Metaphyseal Bone

The increased phosphohydrolytic activity found in calcifying cartilage has breen implicated in the process of normal calcification. Part of this multipotential activity was found to be associated with an extracellular vesicle presumed to be the initial side of calcium salt deposition.
The phosphohydrolytic activity of water extracts from calcifying cartilage and metaphyseal bone has been resolved into three enzymatic entities by DEAE-cellulose column chromatography. The activity which was eluted first, phosphatase I (pyrophosphatase I), increases as cartilage differentiates and calcifies. This increase could serve as a marker for cartilage differentiation and/or calcification. Antibodies to this enzyme isolated from calcifying cartilage or metaphyseal bone cross-react suggesting that the enzymes might, at least in part, be similar.
Cartilage and bone also possess an inorganic pyrophosphatase, pyrophosphatase II, eluted second through the DEAE-cellulose column and another phosphatase, phosphatase II, which was eluted last. By enzymatic and immunologic criteria, it appears that bone and cartilage have the same phosphate-releasing activities indicative of tissues with common cellular origin.
The possible transformation of the differentiating chondrocyte into an osteoblast or osteocyte has been postulated as the cellular mechanism whereby calcified cartilage is replaced by bone. The similarity between the phosphatase I found in epiphyseal cartilage and metaphyseal bone suggests that such transformation is quite likely.     

Purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) has been enriched more extensively than previously from fresh rat liver. For this, 10% homogenates in sucrose-phosphate buffer were treated with 0.1% Tween-20 prior to high-speed centrifugation to obtain soluble proteins. Those precipitated by 40% saturation with ammonium sulfate were subjected to stepwise addition of calcium phosphate gel to remove pyrophosphatase, and the remaining synthetase was further enriched by passage through a tricalcium phosphate column. An apparent yield of greater than 70% and purification over 70-fold was achieved from the high-speed supernatant fraction. The synthetase activity in solution at 4 degrees was largely lost within a week unless protected by thiols which could partly restore inactivated enzyme. The pH optimum for synthetase activity is near 7.7 when assayed with suitable concentrations of FMN, ATP, and Mg2+. Purified enzyme could be separated into lower (140,000) and higher (325,000) molecular weight components when subjected to molecular sieving on a Sephadex G-200 column.     

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.     

In vitro labeling of hydroxyapatite minerals by an engineered protein

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.     

Identification of an extracellular nucleotide pyrophosphatase in the culture media of Streptomyces mediterranei ME/R 17]

An exocellular pyrophosphatase, active on the nucleotide precursors of peptidoglycans, has been found in the culture medium of Streptomyces mediterranei ME/R 17. This enzyme was separated from the DD-carboxypeptidase by batchwise adsorption on DEAE cellulose. The pyrophosphatase had no strict substrate requirements, it hydrolyzed various UDP-sugar substrates: UDP-GlcNAc, UDP-Mur NAc and UDP-MurNAc peptides, giving rise to the corresponding sugar phosphate and to UMP. The enzyme preparation also contained a 5'-nucleotidase activity and UMP was further split to give uridine. This nucleotidase activity was inhibited by potassium tetraborate. Both cytoplasmic and particulate preparations from cells of S. mediterranei also contained a pyrophosphatase activity while only the particulate fractions showed the DD-carboxypeptidase activity. The pyrophosphatase excretion was tested during the grwoth cycle. The activity of the enzyme showed a constant increase throughout the exponential growth and a stronger increase in the late exponential phase. Such a result could be correlated with a consumption of the nutrients in the culture medium, in fact a relatively poor culture medium had a strong positive effect upon the production of the exocellular pyrophosphatase.     

The role of nucleoside triphosphate pyrophosphohydrolase in in vitro nucleoside triphosphate-dependent matrix vesicle calcification

Nucleoside triphosphate pyrophosphohydrolase (EC 3.6.1.8) activity is associated with matrix vesicles purified from collagenase digests of fetal calf epiphyseal cartilage. This enzyme hydrolyzes nucleoside triphosphates to nucleotides and PPi, the latter inducing precipitation in the presence of Ca2+ and Pi. An assay for matrix vesicle nucleoside triphosphate pyrophosphohydrolase is developed using beta, gamma-methylene ATP as substrate. The assay is effective in the presence of matrix vesicle-associated ATPase, pyrophosphatase, and alkaline phosphatase activities. A soluble nucleoside triphosphate pyrophosphohydrolase is obtained from matrix vesicles by treatment with 5 mM sodium deoxycholate. The solubilized enzyme induced the precipitation of calcium phosphate in the presence of ATP, Ca2+, and Pi. Extraction of deoxycholate-solubilized enzymes from matrix vesicles with 1-butanol destroys nucleoside triphosphate pyrophosphohydrolase activity while enhancing the specific activities of ATPase, pyrophosphatase, and alkaline phosphatase. In solutions devoid of ATP and matrix vesicles, concentrations of PPi between 10 and 100 microM induce calcification in mixtures containing initial Ca2+ X P ion products of 3.5 to 7.9 mM2. This finding plus the discovery of nucleoside triphosphate pyrophosphohydrolase in matrix vesicles supports the view that these extracellular organelles induce calcium precipitation by the enzymatic production of PPi. Nucleoside triphosphate pyrophosphohydrolase is more active against pyrimidine nucleoside triphosphates than the corresponding purine derivatives. The pH optimum is 10.0 and the enzyme is neither activated nor inhibited by Mg2+ or Ca2+ ions or mixtures of the two. Vmax at pH 7.5 for beta, gamma-methylene ATP is 0.012 mumol of substrate hydrolyzed per min per mg of protein and Km is below 10 microM. The enzyme is irreversibly destroyed at pH 4 and is stable at pH 10.5.     

Various physico-chemical properties of inorganic pyrophosphatase from the mouse spleen

Manganese, calcium and mercury ions, as well as p-chloromercury benzoate and dithiothreitol are studied for their effect on the activity of inorganic pyrophosphatase (EC 3.6.1.1) of mice spleen. It is shown that Ca2+ and Mn2+ are inhibitors of this enzyme, but Mn2+ in low concentrations may replace Mg2+ in the pyrophosphatase reaction. Hg2+ and p-chloromercury benzoate inhibit the pyrophosphatase activity essentially but not completely. Mice spleen pyrophosphatase is very labile: its preincubation without the substrate for 30 min at 37 degrees C leads to a complete loss of the activity. Neither glycerol, nor glutathione and cysteine but magnesium ions, dithiothreitol and 2-mercaptoethanol protect the enzyme from inactivation. The enzyme is purified by the sulphate ammonium salting-out, gel filtration on Sephadex G-100 as well as by isoelectrofocusing in 5% PAAG. Then pyrophosphatase is eluted from gel and subjected to electrophoresis in the plane layer of the linear gradient of 5-15% PAAG with SDS or 5-25% PAAG without denaturing conditions. One zone corresponding to the molecular mass of 70 kDalton is obtained. It is splitted into two zones in electrophoresis with SDS and 2-mercaptoethanol.     

The roles of annexins and alkaline phosphatase in mineralization process

In this review the roles of specific proteins during the first step of mineralization and nucleation are discussed. Mineralization is initiated inside the extracellular organelles-matrix vesicles (MVs). MVs, containing relatively high concentrations of Ca2+ and inorganic phosphate (Pi), create an optimal environment to induce the formation of hydroxyapatite (HA). Special attention is given to two families of proteins present in MVs, annexins (AnxAs) and tissue-nonspecific alkaline phosphatases (TNAPs). Both families participate in the formation of HA crystals. AnxAs are Ca2+ - and lipid-binding proteins, which are involved in Ca2+ homeostasis in bone cells and in extracellular MVs. AnxAs form calcium ion channels within the membrane of MVs. Although the mechanisms of ion channel formation by AnxAs are not well understood, evidence is provided that acidic pH or GTP contribute to this process. Furthermore, low molecular mass ligands, as vitamin A derivatives, can modulate the activity of MVs by interacting with AnxAs and affecting their expression. AnxAs and other anionic proteins are also involved in the crystal nucleation. The second family of proteins, TNAPs, is associated with Pi homeostasis, and can hydrolyse a variety of phosphate compounds. ATP is released in the extracellular matrix, where it can be hydrolyzed by TNAPs, ATP hydrolases and nucleoside triphosphate (NTP) pyrophosphohydrolases. However, TNAP is probably not responsible for ATP-dependent Ca2+/phosphate complex formation. It can hydrolyse pyrophosphate (PPi), a known inhibitor of HA formation and a byproduct of NTP pyrophosphohydrolases. In this respect, antagonistic activities of TNAPs and NTP pyrophosphohydrolases can regulate the mineralization process.     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.