Disaggregation of hydroxyapatite crystals

A system has been developed to measure quantitatively the disaggregation of hydroxyapatite crystals. Disaggregation was induced by pyrophosphate, ethane-1-hydroxy-1,1-diphosphonate, dichloromethylene diphosphonate, heparin and citrate. Hyaluronic acid stimulated aggregation at low concentrations and disaggregation at high concentrations. Lactate had no effect. The possible role might play in the resorption of calcified tissues in vivo is discussed.     

Disaggregation of hydroxyapatite crystals.

A system has been developed to measure quantitatively the disaggregation of hydroxyapatite crystals. Disaggregation was induced by pyrophosphate, ethane-1-hydroxy-1,1-diphosphonate, dichloromethylene diphosphonate, haparin and citrate. Hyaluronic acid stimulated aggregation at low concentrations and disaggregation at high concentrations. Lactate had no effect. The possible role disaggregation might play in the resorption of calcified tissues in vivo id discussed.     

Raman and infrared studies of homogeneous forms of acid phosphatase from rat liver

Raman spectra of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) forms from rat liver in water solution, and infrared spectra of the same forms as thin films, have been investigated. The spectra show strong bands belonging to phosphodiester or phosphomonoester residues. These groups are modified during the postsynthetic modification of acid phosphatase and are probably connected with the process of bonding and splitting of mannose 6-phosphate and N-acetylglucosamine, in agreement with previous biochemical models for the intracellular transport of newly synthesized lysosomal hydrolases to lysosomes. Some other bands in the infrared spectra are assigned to vibrations of N-H groups which may belong to N-acetylglucosamine.     

Phase and Period Responses of the Two-Peak Circadian Rhythm of Trigonoscelis gigas Reitter (Coleoptera: Tenebrionidae) for 6-hr Light Pulses

Abstract

Enzymatic activity of five lysosomal hydrolases: acid p‐nitrophenyl phosphatase (EC 3.1.3.2), acid β‐glycerophosphatase (EC 3.1.3.2), arylsulphatase (EC 3.1.6.1), β‐galactosidase (EC 3.2.1.23) and β‐N‐acetylhexosaminidase (EC 3.2.1.30) was studied in the supernatants of homogenates of hearts of unirradiated mice, serving as controls, and a group of U.V.‐irradiated mice.

In the control group, determinations made at 6‐hr intervals showed rhythmic diurnal changes in activities of three acid hydrolases. These changes were statistically significant in the case of acid p‐nitrophenyl phosphatase, acid β‐glycerophosphatase, and β‐N‐acetylhexosaminidase. The effect of U.V.‐irradiation was manifested mainly by depression of enzyme activities of the acid hydrolases during the first few hours after exposure. Depression of activities of arylsulphatase and β‐N‐acetylhexosaminidase by U.V. was statistically significant. Presumably, the fall in enzyme activities of the acid hydrolases was due to chemical mediators formed in the skin under the influence of U.V.‐irradiation and adrenal corticoids secreted into the blood.     

Differences in induction of lysosomal protease activity by protease inhibitors in B16 melanoma cell lines

1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction.     

An isoelectric focusing study of acid phosphatase heterogeneity in rat liver

The different forms of acid phosphatase (EC 3.1.3.2) in rat liver homogenates, lysosomal, mitochondrial, microsomal fractions and cytosol were studied with isoelectric focusing. Evidence is presented that isoelectric focusing of acid phosphatase in subcellular fractions shows individual changes and time related patterns. Mild autolysis shifted all enzyme activity peaks of isoelectric focusing patterns to the one at pH 7.04.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.     

Fasciola hepatica: Histochemical observations on juveniles and adults and the cytopathological changes induced in infected mouse liver

The histochemical characteristics of juvenile intrahepatic forms of Fasciola hepatica have been compared with those of the adults. The histochemical distributions of carboxylesterase (EC 3.1.1.1), acetylcholinesterase (EC 3.1.1.7) and alkaline phosphatase (EC 3.1.3.1) were similar in both juvenile and adult forms. Differences were apparent in occurrence and distribution of β-glucuronidase (EC 3.2.1.31) and N-acetyl-β-glucosaminidase (EC 3.2.1.29) activities in the tegument, parenchyma, and caecal cells. These may reflect the dietary mode and relationship to the host of the juvenile intrahepatic and adult bile-duct forms. Starvation of both juveniles and adults is accompanied by an increase in the granule-associated staining reactions for acid hydrolases in some of the parenchymal cells with a concomitant decrease in staining for glycogen in these cells. This response to starvation is reversible with refeeding, indicating that it is probably a genuine response to nutrient stress and not to degeneration induced by the in vitro conditions of the flukes. Carboxylesterase and acid phosphatase (EC 3.1.3.2) have been demonstrated to be polymorphic, using polyacrylamide disc electrophoresis. Changes were observed in the staining patterns with starvation. The juvenile flukes stimulated a considerable increase in the level of lysosomal hydrolases and alkaline phosphatase in mouse hepatic cells.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture.

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.     

Role of ATP and enzyme-bound nascent peptides in the control of elongation for mycobacillin synthesis.

The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+-dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues.     

Repression of the acid phosphatase of Saccharomyces bisporus in relation to the polyphosphate content of the cells.

The relationship between the level of stored polyphosphate in growing cells of Saccharomyces bisporus and the repressing or derepression of the synthesis of the enzyme acid phosphatase (EC 3.1.3.2) was investigated. Time-course studies showed that there is no correlation between the cellular concentrations of either polyphosphate or orthophosphate and the ability of the cells to form this enzyme. The only compound investigated that was capable of repressing acid phosphatase synthesis was orthophosphate in the growth medium (i.e. orthophosphate outside the cell).     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

ON THE MECHANISMS OF BONE RESORPTION : The Action of Parathyroid Hormone on the Excretion and Synthesis of Lysosomal Enzymes and on the Extracellular Release of Acid by Bone Cells

Bone resorption, characterized by the solubilization of both the mineral and the organic components of the osseous matrix, was obtained in tissue culture under the action of parathyroid hormone (PTH). It was accompanied by the excretion of six lysosomal acid hydrolases, which was in good correlation with the progress of the resorption evaluated by the release of phosphate, calcium 45 or hydroxyproline from the explants; there was no increased excretion of two nonlysosomal enzymes, alkaline phosphatase, and catalase. Balance studies and experiments with inhibitors of protein synthesis indicated that the intracellular stores of the acid hydrolases excreted were maintained by new synthesis. The release was not due to a direct disruption of the lysosomal membrane by PTH; it is presumed to result from an exocytosis of the whole lysosomal content and to involve mechanisms similar to those controlling the secretion of this content into digestive vacuoles. The resorbing explants acidified their culture fluids at a faster rate and released more lactate and citrate than the controls; this release was in good correlation, in the PTH-treated cultures, with the resorption of the bone mineral, but the amount of citrate released was considerably smaller than that of lactate. The acid released could account for the resorption of the mineral. It is proposed, as a working hypothesis, that the acid hydrolases of the lysosomes are active in the resorption of the organic matrix of bone and that acid, originating possibly from the stimulation of glycolysis, cares for the concomitant solubilization of bone mineral while also favoring the hydrolytic action of the lysosomal enzymes.     

Enzymes and proteins in bile. Variations in output in rat cannula bile during and after depletion of the bile-salt pool.

The protein concentration in bile from several species is reported. The changes in output of protein, bile salts and several enzymes have been followed in rat bile over a 48 h cannulation period. Bile-salt concentration dropped rapidly owing to interruption of the enterohepatic circulation but the output of protein, lysosomal enzymes [acid phosphatase (EC 3.1.3.2) and beta-D-glucuronidase (EC 3.2.1.31)] and plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I (EC 3.1.4.1)] was maintained. Liver cell damage, monitored by output of lactate dehydrogenase, was very low throughout. Protein, lysosomal enzymes and plasma-membrane enzymes showed different patterns of output with time, but all showed a net increase between 12 and 24 h. The output of lysosomal and plasma-membrane enzymes was between 1 and 5% of the total liver complement over the first 24 h; if inhibition by biliary components is taken into account the output of some of these enzymes, particularly acid phosphatase, may be greater. Ultracentrifugation of bile showed that as the concentration of bile salts decreases the proportion of plasma-membrane enzymes in a sedimentable form increases. The results are discussed in relation to other studies of biliary proteins and to studies of the perturbation of membranes and cells with bile salts.     

Dimeric nature and amino acid compositions of homogeneous canine prostatic human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r     

High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates

The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.