Elution of Acid Phosphatase from the Cell Surface of Saccharomyces mellis by Potassium Chloride.

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of acid phosphatase from the cell surface of Saccharomyces mellis by potassium chloride. J. Bacteriol. 90:82-94. 1965.-Acid phosphatase of Saccharomyces mellis may be eluted from intact resting cells by 0.5 m KCl or other salts. However, the enzyme is not eluted at higher salt concentrations of about 2 m unless a thiol, such as beta-mercaptoethanol, is included in the reaction mixture. These treatments do not significantly affect viability of the cells. Neutral compounds like sorbitol or sucrose cannot substitute for ionic compounds in eluting the enzyme from resting cells. Furthermore, the neutral compounds are also inadequate for stabilizing the protoplast structure. It is suggested that the enzyme is held on the cell surface by a combination of electrostatic forces and disulfide bonds. Thiol alone dissociates protein and carbohydrate from the cell surface, but the eluate has no acid phosphatase activity. Salts also remove protein and carbohydrate from the cell surface, but the amount of protein removed is considerably less than that dissociated by thiol. A concentration of 0.5 m KCl elutes more protein than does a 2 m concentration, and enzymatic activity is present only in the 0.5 m KCl eluate. The carbohydrate eluted by either reagent has been identified as a mannan. Conditions for eluting acid phosphatase from acetonedried cells of S. mellis are essentially the same as those for resting cells. Significantly, though, thiol is required at all salt concentrations to dissociate the enzyme. Pretreatment of the cells with thiol, followed by KCl, elutes acid phosphatase, whereas the reverse procedure does not. Acid phosphatase is excreted by growing cells of S. mellis into growth media if the medium contains 0.25 m KCl. The total yield of enzymatic activity may be 8 to 10 times greater than is usually present on derepressed cells grown in a salt-free medium. The enzyme can be precipitated from the culture fluid with acetone. The acetone-precipitated fraction contains mannan and protein in a ratio of 12:1 by weight. Partial purification of the enzyme by calcium phosphate gel and elution resulted in an enzyme fraction in which the specific activity on the basis of protein increased 12-fold, and the carbohydrate-protein ratio was reduced to 1:1.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Recovery of exocellular acid phosphatase activity on Saccharomyces mellis after treatment of the organism with reagents that affect the cell surface

Derepressed cells of Saccharomyces mellis were treated in one of several different ways to either elute or inactivate the exocellular enzyme, acid phosphatase. The enzyme was either (i) eluted from resting cells with 0.5 m KCl plus 0.1% beta-mercaptoethanol, (ii) eluted from exponential phase cells by growing the organism in derepressing media containing 0.5 m KCl, or (iii) inactivated on exponential phase cells by adding sufficient acid or base to growth media to destroy the enzyme but not enough to kill the cells. These treatments did not affect viability. Treated cells were transferred to fresh growth media or some other reaction mixture, and the kinetics of recovery of acid phosphatase activity was studied. In these reaction mixtures, enzyme was synthesized only by actively growing cells. Treated resting cells were indistinguishable from untreated, repressed resting cells in that the organism inoculated into complete growth medium remained in the lag phase for approximately 6 hr before both growth and enzyme synthesis began. Exponential phase derepressed cells treated by method (ii) or (iii) were transferred to fresh medium under conditions that allowed growth to continue. The cells immediately started to manufacture enzyme at a rate greater than normal until the steady-state level was reached, thus demonstrating a feedback control system. Exponential phase repressed cells were also transferred to fresh derepressing media under conditions which sustained growth. Though these cells began to grow immediately, there was a lag before acid phosphatase synthesis began followed by a lengthy inductive period. The length of the period of induction could be correlated with the polyphosphate content of the cells. As the supply of polyphosphate neared exhaustion, the rate of synthesis increased rapidly until it was greater than normal; this differential rate was sustained until the steady-state concentration was reached. When derepressed cells grow in a medium containing 0.5 m KCl, some acid phosphatase activity is found free in the culture fluid and some remains firmly attached to the cells despite the presence of the salt. The bound activity is subject to feedback control, but the steady-state level of this activity on the cells is only one-third that of the acid phosphatase on cells growing in nonsaline media. The extracellular phosphatase is produced at a rate that is several-fold greater than that of the exocellular enzyme in a nonsaline medium. The synthesis of the extracellular enzyme does not seem to be controlled by a feedback mechanism but is produced at a maximal rate as long as the cells are growing.     

Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme

Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000.     

Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria.

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.     

Affinity separation of an acid phosphatase from rat tissues and Gaucher spleen with immobilized Cibacron Blue

An acid phosphatase species which was activated by Fe2+ was determined to be partially soluble but mainly particulate in rat spleen. The particulate enzyme could be extracted into 1 M KCl. This enzyme bound to Cibacron Blue-immobilized Sepharose (Blue-Sepharose) and was desorbed by 2 M KCl with a good yield, while the other acid phosphatases in rat spleen did not adsorb on Blue-Sepharose. The enzymes eluted on Blue-Sepharose chromatography of both the soluble and particulate fractions were electrophoretically identical. The enzyme hydrolyzed aryl monophosphates, phosphoproteins, and nucleoside di- and triphosphates. The activity for the three kinds of substrate was similarly activated by Fe2+, ascorbic acid and cysteine, and inhibited by molybdate, Cu2+ and F-. Cibacron Blue inhibited the enzyme competitively with respect to a substrate, p-nitrophenyl phosphate, but kinetic analysis suggested that more than one dye molecule binds to the enzyme. The Blue-Sepharose technique could be applied not only to quantitative separation of acid phosphatases similar to the spleen enzyme from bone and epidermis of rat, but also to that of a tartrate-resistant acid phosphatase from human spleen with Gaucher's disease.     

Ultrastructural demonstration of phosphatases by perfusion fixation followed by perfusion incubation of rat liver.

An alternative to previous methods (tissue chopper, frozen sections) for the ultrastructural demonstration of phosphatases is described. The present approach is based on a short vascular perfusion of rat liver with glutaraldehyde through the inferior caval vein, followed by vascular perfusion incubation with a medium containing the enzyme substrates. The effect of glutaraldehyde on three different types of phosphatases was investigated, namely a lysosomal enzyme (acid phosphatase) a tightly bound microsomal enzyme (G6Pase) and a loosely bound microsomal enzyme (IDPase). It is demonstrated that by perfusion with glutaraldehyde for three minutes good cellular morphology is obtained and that 50-60% of the initial activity of glucose-6-phosphatase, inosine-diphosphatase and acid phosphatase remains. The localization and deposition of G6Pase activity were distinct and observed throughout the endoplasmic reticulum and the nuclear envelope. For acid phosphatase, the reaction product was confined to various types of lysosomes including presumed autophagic vacuoles. No signs of enzyme diffusion were noted. The present approach seems to offer some advantages: it is simple and requires no extra equipment, penetration of the fixative and incubation enzyme medium is good, and finally freeze artifacts are avoided.     

Purification and physicochemical characterization of a human placental acid phosphatase possessing phosphotyrosyl protein phosphatase activity

A 17-kilodalton (kDa) human placental acid phosphatase was purified 21,400-fold to homogeneity. The enzyme has an isoelectric point of pH 7.2 and a specific activity of 106 mumol min-1 mg-1 using p-nitrophenyl phosphate as a substrate at pH 5 and 37 degrees C. This placental acid phosphatase showed activity toward phosphotyrosine and toward phosphotyrosyl proteins. The pH optima of the enzyme with phosphotyrosine and with phosphotyrosyl band 3 (from human red cells) were between pH 5 and 6 and pH 5 and 7, respectively. The Km for phosphotyrosine was 1.6 mM at pH 5 and 37 degrees C. Phosphotyrosine phosphatase activity was not inhibited by tartrate or fluoride, but vanadate, molybdate, and zinc ions acted as strong inhibitors. Enzyme activity was also inhibited by DNA, but RNA was not inhibitory. It is a hydrophobic nonglycoprotein containing approximately 20% hydrophobic amino acids. The average hydrophobicity was calculated to be 903 cal/mol. The absorption coefficient at 280 nm, E1% 1cm, was determined to be 5.7. The optical ellipticity of the enzyme at 222 nm was -5200 deg cm2 dmol-1, which would correspond to a low helical content. Free sulfhydryl and histidine residues were necessary for the enzyme activity. The enzyme contained four reactive sulfhydryl groups. Chemical modification of the sulfhydryls with iodoacetate resulted in unfolding of the protein molecule as detected by fluorescence emission spectroscopy. Antisera against both the native and the denatured protein were able to immunoprecipitate the native enzyme. However, upon denaturation, the acid phosphatase lost about 70% of the antigenic determinants. Both antisera cross-reacted with a single 17-kDa polypeptide on immunoblotting.     

Alkaline and acid phosphatases from the extensively halotolerant bacteriumHalomonas elongata

Alkaline and acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2, respectively) ofHalomonas elongata were cytochemically localized on the cell envelope. These enzymes were then isolated and partially purified by sonication, ammonium sulfate precipitation, and column chromatography from cells grown in alanine defined medium at 0.05, 1.37, and 3.4M NaCl. Enzyme assays were conducted at pH 5.0 and 9.0 with varying concentrations of NaCl, KCl, and LiCl in the assay buffer. Results showed higher acid phosphatase activity compared with that of alkaline phosphatase; and all enzyme activities were optimal at NaCl concentrations similar to the medium NaCl concentrations for the cells grown at 1.37 and 3.4M. However, minimum enzyme activities were observed for cells grown at the low salt concentration (0.05M). Although samples showed strong activities at some KCl concentrations, generally the enzyme activities decreased significantly when KCl or LiCl was substituted for NaCl. Polyacrylamide gel electrophoresis followed by histochemical staining for the phosphatases showed only one band for both enzymes for each cell sample grown at the different NaCl concentrations.     

Alkaline phosphatase in HeLa cells. Stimulation by phospholipase A2 and lysophosphatidylcholine.

Treatment of homogenates and plasma membrane preparations from HeLa cells with phospholipase A2 (EC 3.1.1.4) caused a 50% increase in activity of membrane-associated alkaline phosphatase. Lysophosphatidylcholine, dispersed in 0.15 M KCl, affected alkaline phosphatase in a similar fashion by releasing the enzyme from particulate fractions into the incubation medium and by elevating its specific activity. Higher concentrations of lysophosphatidylcholine solubilized additional protein from particulate fractions but did not further increase the specific activity of the released alkaline phosphatase. Particulate fractions from HeLa cells were exposed to the effects of liposomes prepared from lysophosphatidylcholine and cholesterol. The ratio of particulate protein/lysophosphatidylcholine (by weight) required for optimal activation of alkaline phosphatase was one. Kinetic studies indicated that phospholipase A2 and lysophosphatidylcholine enhanced the apparent V of the enzyme but did not significantly alter its apparent Km. The increased release of alkaline phosphatase from the particulate matrix by lysophosphatidylcholine was confirmed by disc electrophoresis. The release of the enzyme by either phospholipase A2 or by lysophosphatidylcholine appeared to be followed by the formation of micelles that contained lysophosphatidylcholine. The new complexes had relatively less cholesterol and more lysophosphatidylcholine than the native membranes. The possibility that lysophosphatidylcholine formed a lipoprotein complex with the solubilized alkaline phosphatase was indicated by a break point in the Arrhenius plot which was evident only in the lysophosphatidylcholine-solubilized enzyme but could not be demonstrated in alkaline phosphatase that had been released with 0.15 M KCl alone.     

Evidence of acid phosphatase in the cytoplasm as a distinct entity

A study of subcellular acid phosphatase distribution in mammalian tissues shows that isozymes with specific functions are compartmentalized in the cells. The enzyme may be generalized into two types: type A and type B. They are shown by several means to be distinct entities. Type A is confined to the cytoplasm and is inhibited by Cu2+, HCHO, and the coupling agents (for enzyme staining) fast blue RR salt and fast Garnet GBC salt (newly discovered inhibitors), but is insensitive to fluoride and L-(+)-tartrate. Type B is localized in the organelles, presumably lysosomes, in both soluble form and membrane-bound form, with inhibitor sensitivity exactly opposite to that of type A enzyme. Types A and B consist of different sets of isozymes, with sensitivities to inhibitors resembling those observed with the crude extracts of subcellular fractions. Acid phosphatase that exhibits a phosphoryl transfer property was identified as type A enzyme. Type A enzyme has a slightly higher optimal pH and is inhibited by alloxan, whereas for type B, the addition of alloxan broadens the optimal pH to a higher range and elevates the activity of pH 7.4 from negligible to about 30-40% of that obtained under optimal conditions. The alloxan-mediated elevation of type B enzyme activity to this level at the physiological pH may be of considerable significance. Type B enzyme has a high affinity for metabolic intermediates and nucleotides, while type A has an extremely low affinity for these substrates. Cytoplasmic acid phosphatase (type A) is a significant enzyme population and its activity is not related to the lysosome density in the cells. Type A enzyme in the cytoplasm is thus shown to be an entity distinctly different from type B enzyme in the lysosomes. These findings suggest that the physiological functions of type A acid phosphatase, such as metabolic regulatory processes, merit further studies because of the phosphoryl transfer activity and cytoplasmic localization of the enzyme.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

The modulator protein dissociates the catalytic subunit of hepatic protein phosphatase G from glycogen.

1. The phosphorylase phosphatase and glycogen-synthase phosphatase activities associated with the glycogen particles from rat liver were progressively inhibited by incubation with modulator protein. However, the phosphorylase phosphatase activity of the catalytic subunit was entirely recovered after destruction of the modulator and the regulatory subunit(s) by trypsin. 2. Inhibition of protein phosphatase G by modulator was associated with a translocation of the phosphorylase phosphatase activity (measured after incubation with trypsin) from glycogen to the soluble fraction. The degree of inhibition of phosphatase G corresponded closely to the extent to which the phosphorylase phosphatase activity was released from the glycogen particles. Incubation of glycogen-free protein phosphatase G with modulator did not change the affinity of the enzyme for added glycogen, but decreased the amount of phosphatase that could be bound to glycogen. 3. The phosphorylase phosphatase activity that was released from the glycogen particles by modulator migrated on gel filtration as a complex (Mr 106,000) of the catalytic subunit with modulator. Phosphorylase phosphatase activity could be transferred from glycogen-bound protein phosphatase G to modulator that was covalently bound to Sepharose. After elution from the column, the enzyme was identified as the free catalytic subunit (Mr 37,000).     

The subcellular distribution of triphosphoinositide phosphomonoesterase in guinea-pig brain

1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.     

Affinity chromatography of Ankistrodesmus braunii nitrate reductase using blue dextran-sepharose (author's transl)

Blue Dextran has been coupled covalently to Sepharose-4B to purify the enzymatic complex NAD(P)H-nitrate reductase (EC 1.6.6.2) from the green alga Ankistrodesmus braunii by affinity chromatography. The optimum conditions for the accomplishment of the chromatographic process have been determined. The adsorption of nitrate reductase on Blue Dextran Sepharose is optimum when a phosphate buffer of low ionic strength and pH 6.5-7.0 is used. Once the enzyme has been bound to Blue Dextran Sepharose, it can be specifically eluted by addition of NADH and FAD to the washing buffer. However, none of the nucleotides added separately is able to promote the elution of the enzyme from the column. The elution can be also achieved, but not specifically, by increasing the ionic strength of the buffer with KCl. These results have made possible a procedure for the purification of A. braunii nitrate reductase which led to electrophoretic homogeneity, with an overall yield of 70% and a specific activity of 49 units/mg of protein.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver

A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A-0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used.     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Fractionation of nonhistone proeins on a column of daunomycin-CH-Sepharose 4B

The molecular weight of a partially purified alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) from the halotolerant yeast Debaryomyces hansenii was estimated to 110,000 by gel filtration. The isoelectric point determined by electrofocusing was at approximately pH 4.4. The enzyme had a broad specificity against phosphomonoesters and also attacked some acid anhydrides. Arsenate, molybdate, and orthophosphate acted as competitive inhibitors. Various metal-binding agents inhibited enzyme activity. A zinc addition almost completely reversed the EDTA inhibition. Magnesium stimulated enzyme activity and was required for maintenance of activity at high concentrations of Na+. Increasing glycerol concentration increased the value of the Michaelis constant (Km) and decreased the maximum velocity (V). Solutions equimolar in KCl and NaCl stimulated enzyme activity by increasing V, whereas the Km was almost unaffected by salt concentration. Enzyme extracted from cells cultured at low salinity was indistinguishable from that of cells grown in the presence of 2.7 M NaCl with respect to several criteria.     

Acid phosphatase of HeLa cells: properties and regulation of lysosomal activity by serum.

Although the subcellular distribution profile of acid phosphatase in HeLa cells is typical of a lysosomal enzyme, different lysosomal (70–80%) and supernatant forms (20–30%) have been demonstrated by their differences in pH activity curves, substrate specificities, thermal stability, sensitivity to inhibitors, and kinetics. Enzymes of the lysosomal fraction displayed anomalous kinetics in the hydrolysis of p-nitrophenyl phosphate. The major lysosomal acid phosphatase activity appears to be associated with the membrane.The total acid phosphatase activity in the cell is controlled by the concentration of serum in the medium. The specific activity in the homogenates of cells grown in high serum concentration (30%) is about twice that of cells grown in low serum concentration (1%). This doubling of specific activity holds for the lysosomal enzyme (or enzymes), but little change occurs in the supernatant form (or forms). Two other lysosomal enzymes, β-glucuronidase and N-acetyl-β-d-hexosaminidase, do not increase in specific activity. The serum-dependent formation of acid phosphatase is sensitive to cycloheximide, actinomycin D, and cordycepin. Cycloheximide blocks the increase in enzymatic activity immediately, whereas cordycepin and actinomycin D have no effect for at least 8 h. These findings suggest that de novo protein synthesis is involved in the induction of lysosomal acid phosphatase by serum and that the mRNA for this enzyme is relatively stable.