A comparative study of microsomal and cytosolic S6 phosphatase activities in rat liver

Summary Spontaneous S6 phosphatase activities dephosphorylating Ser(P)-235 and Ser(P)-236 of the ribosomal protein S6 were measured and compared in microsomes and cytosol of rat liver. The substrate used, small (40S) ribosomal subunits ³²P-labelled in vitro by protein kinase A, contained phosphorylated S6 (mainly in the dephosphorylated form) and some minor phosphorylated species. The microsomal and cytosolic S6 phosphatase activities displayed a number of distinct properties. The microsomal activity, representing ca 20% of the S6 phosphatase activity in the post-mitochondrial supernatant, was mainly due to a type-1 phosphatase and dephosphorylated only S6. The remaining post-mitochondrial S6 phosphatase activity, which was fully recovered in the cytosol, and appeared to result from a combination of type-1 (43%) and type 2 (57%) phosphatases, acted on S6 as well as on the minor phosphorylated species. The microsomal activity was 50% inhibited by MgCl₂ (l0 mM) and was stimulated at least 4.3 fold by MnCl₂ (1 mM), while the cytosolic activity was inhibited only 18% by Mg²⁺ (10 mM) and was increased 2.2 fold by Mn²⁺ (1 mM). The microsomal activity was increased 10% (P < 0.06) by lower doses of insulin (25 U/Kg) and 14% (P < 0.05) by vanadate, but was not significantly (P > 0.10) affected by larger doses of insulin (100 U/kg), hepatectomy or cycloheximide. By comparison the cytosolic S6 phosphatase activity was unresponsive to insulin and vanadate, but was decreased 14% and 17% (P < 0.05) by hepatectomy and cycloheximide. It is concluded that (i) there. are clear differences between the microsomal and cytosolic S6 phosphatase activities, which may be relevant to their specific functions in the cell, and (ii) the inhibition of cytosolic S6 phosphatase activity by hepatectomy and cycloheximide may contribute to the increase in hepatic S6 phosphorylation induced by these treatments.     

Reversible inhibition of skeletal muscle phosphoprotein phosphatase by ATP, phosphate and fluoride.

A phosphoprotein phosphatase preparation which showed activity towards glycogen synthase, phosphorylase, phosphorylase kinase, and phosphohistones was reversibly inhibited (70–90%) by preincubation with free ATP (apparent K_i about 0.3 mM). Other nucleotides (ADP2 (apparent K_a 3μM) prior to assay. Other divalent metals (Co⁺⁺ > Zn⁺⁺ > Mg⁺⁺) were partially effective in reversing the inhibition. It is concluded that ATP by virtue of its special structure and metal binding capacity possibly removes a catalytically important metal ion from the enzyme.     

Starvation reduces pyruvate dehydrogenase phosphate phosphatase activity in rat kidney

Pyruvate dehydrogenase complex (PDC) from rat kidney or pig heart previously inactivated by phosphorylation (PDHP) was activatedin vitro by PDHP phosphatase from kidneys of starved or fed rats. Starvation for 48 h of the rats from which the PDC was prepared led to a decrease in the rate of activation of PDC at early time periods (<2 min), particularly at submaximal concentrations of Mg²⁺. Using intact permeable kidney mitochondria incubated for 15 sec, it was found that starvation of rats more than doubled the Mg²⁺ concentration at which the half maximal increment of PDC activity (PDC_a) was observed. Reduction of PDHP phosphatase activity due to starvation was also apparent when phosphatase was separated from PDC and recombined with PDC from the same or different animals.

Intraperitoneal injection of insulin and glucose 1 h before sacrifice of starved rats prevented the reduction of PDHP phosphatase activity whether or not protein synthesis was inhibited. The effect of insulin in restoration of PDHP phosphatase activity of starved rats was not mimicked by 5-methylpyrazole 3-carboxylic acid, an inhibitor of lipolysis.

When renal PDHP phosphatase was incubated with pig heart PDC in the presence of 10 mM Mg²⁺ and 0.1 mM Ca²⁺ the increment in PDC_a, in 1 min was 30% of fully activated PDC activity (PDC₁) observed after 15 min. Removal of divalent cations did not affect the increment in 1 min but prevented further increments. Conversely okadaic acid diminished 1 min increment but did not disturb PDC_t. It is suggested that the different behaviour of renal PDC from fed and starved animals may partly be due to different divalent cation independent PDHP phosphatase activity.

     

The K+- and HCO3−-stimulated phosphatase activities in renal microsomes of rats

The K⁺-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO₃^?-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO₃^?-stimulated phosphatase activity, but did not enhance the K⁺-stimulated phosphatase activity. Ouabain did not inhibit the HCO₃^?-stimulated phosphatase activity, but inhibited the K⁺-stimulated phosphatase activity.The renal K⁺-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO₃^?-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K⁺-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na⁺ + K⁺)-ATPase activity by aldosterone treatment (P < 0.02).     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Phosphatase activity in cultured glioma and neuroblastoma cells

Acid phosphatase activity in human glioma cells (138 MG) and mouse neuroblastoma cells (C 1300) was associated with structures accumulating neutral red and acridine orange. Only neuroblastoma cells gave a significant positive histochemical reaction for alkaline phosphatase. Glioma and neuroblastoma cell homogenates exhibited maximal phosphatase activity at pH 5 as measured by spectrophotometer. The specific activity; μmoles phosphate released per hour/mg protein was 1.1 in glioma and 0.9 in neuroblastoma. At pH 8, glioma cells lacked activity whereas neuroblastoma cells showed another maximum. The acid phosphatase activity of both cell types was strongly inhibited by CuCl₂ (0.3 mM) and NaF (10 mM) and moderately by -tartaric acid (10 mM). cGMP (1 mM) stimulated the phosphatase activity of both cell lines. db-cAMP, in serum-free medium, induced characteristic morphological changes of the cells studied. This process was unaffected by CuCl₂, c-GMP and -tartaric acid. db-cAMP (1 mM) inhibited proliferation in both glioma and neuroblastoma cells during a 48 h incubation in serum-containing medium. This growth inhibition was associated with an increase in acid phosphatase activity of the glioma but not of the neuroblastoma cells.     

Alkaline phosphatase activities in muscle of the euryhaline crab Chasmagnathus granulatus: Response to environmental salinity

The occurrence, characteristics and response to environmental salinity of alkaline phosphatase (AP) activity were studied in chela muscle of the euryhaline crab Chasmagnathus granulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Chela muscle exhibited a levamisole-insensitive and a levamisole-sensitive AP activities with distinct characteristics. Levamisole-insensitive activity appeared to be maximal at pH 7.7, whereas levamisole-sensitive AP activity was similar with the range of pH 7.4 to 8.0. Both activities at pH 7.7 exhibited a Michaelis-Menten kinetics (K_m = 0.789 and 1.416 mM, respectively). I₅₀ for levamisole-sensitive AP activity was about 12 mM. Levamisole-insensitive and levamisole-sensitive AP activities were differentially affected by temperature. Levamisole-sensitive AP activity was quite sensitive to temperature, exhibiting a peak at 37 °C but being low at 5 to 30 °C and 45 to 60 °C. Both activities were inhibited by Cu²⁺. At 1.0 mM Cu²⁺, levamisole-insensitive AP activity was inhibited about 82% whereas levamisole-sensitive AP activity was almost completely inhibited. Levamisole-insensitive AP activity appeared to be sensitive to environmental salinity. In crabs acclimated to low salinity (10‰) this activity was lower than in 35‰ salinity. The response to environmental salinity suggests that levamisole-insensitive AP activity could be a component of muscle regulatory mechanisms at the biochemical level secondary to hyperregulation of C. granulatus. The possible physiological roles and functional relationship of AP activity with Na⁺/K⁺ ATPase in muscle are discussed.     

Acid Metallophosphatase from the Ameba Amoeba proteus

Tartrate-resistant acid phosphatase (TR-AcPh) from the ameba Amoeba proteus is represented by 3 bands (electromorphs) revealed after disk-electrophoresis in PAAG, using 2-naphthylphosphate as substrate. The presence of 50 mmol/l MgCl₂ or CaCl₂ in the incubation mixture increases activities of all electromorphs of TR-AcPh, while of ZnCl₂, of two of them. The activity of the TR-AcPh electromorphs also rose after the 30-min incubation of the gels in MgCl₂, CaCl₂ or ZnCl₂ (10 and 100 mM) before gel staining. However, 1 M ZnCl₂, unlike 1 M CaCl₂ or 1 M MgCl₂, partly inactivated two out of three TR-AcPh electromorphs. The TR-AcPh electromorphs were inhibited by 1,10-phenanthroline (1,10-Ph), EDTA, and EGTA (all at a concentration of 5 mM) faster than by H₂O₂ (10 mM). The inactivation of the TR-AcPh electromorphs by the chelating agents did not depend (EGTA) or nearly did not depend (EDTA, 1,10-Ph) on their concentration (0.05, 0.5, and 5 mM). Out of 5 tested ions (Mg²⁺, Ca²⁺, Fe²⁺, Fe³⁺, and Zn²⁺), only Zn ions reactivated the TR-AcPh electromorphs inactivated by 1,10-Ph, EDTA or EGTA. The TR-AcPh electromorphs were reactivated worse after inactivation by EGTA than by EDTA or 1,10-Ph. It is suggested that the active site of TR-AcPh contains the zinc ion essential for catalytic activity of this enzyme, i.e., TR-AcPh of A. proteus is a metallophosphatase performing the phosphomonoesterase activity in acidic medium.     

Purification and characteristics of a specific alkaline phosphatase from the integument of the mediterranean fruit fly,Ceratitis capitata

A specific alkaline phosphatase (ALPase) from the integument of white pupae has been purified 500-fold. The purification procedure included solubilization with Triton X-100, butanol extraction, fractionation with ammonium sulfate, and chromatography on concanavalin A-Sepharose, Sephadex G-200, and Sepharose 6B. Two peaks with enzyme activity were observed. The major peak had a molecular weight of approximately 180,000, while the minor peak, which had identical kinetic parameters and substrate specificity as those of the major one, was eluted in a high molecular weight form (about 900,000), probably cross-linked with chitin, since the enzyme was separated from the chitin only by lysozyme treatment. The enzyme hydrolyzes only tyrosine phosphate and β-glycerophosphate, with apparent K_ms of 0.35 mM and 0.22 mM, respectively, but not serine phosphate, threonine phosphate, ATP, and AMP. The optimum pH was in the alkaline range, with a peak at pH 9.4. The divalent cations Mn²⁺, Mg²⁺, and Ba²⁺ had stimulatory actions, while Cu²⁺ exerted a very strong inhibitory action on the enzyme activity. The ALPase was inhibited by L-tyrosine in a dose-dependent fashion. At a concentration of 2 mM, L-tyrosine totally inhibited the enzyme activity, while L-phenylalanine inactivated the enzyme about 25%. The accumulated evidence that ALPase is involved in the sclerotization process of insect integument is discussed.     

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using ¹²⁵I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5′-nucleotidase, Mg²⁺-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K⁺ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-γ-³²P in the presence of MgCl₂ was of high specific activity and was optimum at pH 7.0 and 8.2. K⁺ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg²⁺-ATPase activity was inhibited by ouabain in the presence of Na⁺ and K⁺. Since K⁺-stimulated phosphatase activity does not correlate with Mg²⁺-ATPase, the two assay systems define separate enzymatic processes.     

Delta endotoxin inhibits a phosphatase in midgut epithelial membranes of Heliothis virescens

A vanadate- and delta endotoxin-sensitive phosphatase copurified with plasma membranes and brush border membranes from the midgut epithelium of Heliothis virescens. Phosphatase activity was stimulated under alkaline conditions. Total phosphatase activity was inhibited 60% in the midgut membranes by 360 nM delta endotoxin with a K_i = 76 nM. Brush border membranes were inhibited 75% by 1.47 μM delta endotoxin with a K_i = 64.7 nM. Vanadate (200 μM) completely inhibited the toxinsensitive alkaline phosphatase. A 72 kDa protein was phosphorylated when membranes were incubated with Mg²⁺ and [³²P]orthophosphate, and phosphorylation was inhibited by both vanadate and delta endotoxin, suggesting that destabilization of this phosphoprotein was responsible for phosphatase inhibition.     

Alkaline phosphatase activity sensitive to environmental salinity and dopamine in muscle of the euryhaline crab Cyrtograpsus angulatus

The occurrence, characteristics and response to environmental salinity and dopamine of alkaline phosphatase (AP) activity were studied in chela muscle of the euryhaline crab Cyrtograpsus angulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Chela muscle exhibited a high AP activity with a Michaelis-Menten kinetic (K_m=1.21 mM). AP activity was strongly inhibited by EDTA (I₅₀=2.26 mM). AP activity appeared to be sensitive to environmental salinity. In crabs acclimated to low salinity (10‰) AP activity was lower than in 35‰ salinity. Upon an abrupt change to reduced salinity a short-term decrease of AP activity occurred, concomitant with the transition to hyperregulation. Furthermore, AP activity appeared to be under hormonal control since it was inhibited “in vivo” by 10⁻⁴ M dopamine. The response to both environmental salinity and dopamine suggests that AP activity could be a component of muscle regulatory mechanisms at the biochemical level secondary to hyperregulation of C. angulatus. The possible functional relationship of AP activity with Na⁺/K⁺ ATPase in muscle is discussed.     

The effect of amino acids,monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

Summary The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-¹⁴C] pyruvate in the presence of 0.05 mM Ca²⁺ and Mg²⁺. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(-morpholinopropionyl) benzo[b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-¹⁴C] pyruvate to release ¹⁴CO₂. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg²⁺ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg²⁺. However, at Mg²⁺ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.     

Characterization of phospholipase A2 and acyltransferase activities in squid (loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Effect of extraneous zinc on calf intestinal alkaline phosphatase

The effect of extraneous zinc on calf intestinal alkaline phosphatase was studied for quick reversible binding and slow irreversible binding of zinc ions at various concentrations. Under the conditions of slow binding of zinc to CIP increasing Zn²⁺ (less than 1.0 mM, n_M/n_E 1.0 × 10⁶) inhibited enzymatic activity, and further increasing Zn²⁺ resulted in an increase of activity. For quick reversible binding of Zn²⁺, the effect on CIP activity changed at lower concentrations of substrate, indicating a complex cooperativity between Zn²⁺ and pNPP. Both protein intrinsic emission fluorescence and ANS-bound protein fluorescence, as well as circular dichroism spectra have shown that the binding of zinc ions changed the enzyme conformation, which was the reason for the changes in enzyme activity induced by extraneous zinc.     

Purification and characterization of extracellular acid phosphatase of Tetrahymena pyriformis

An extracellular acid phosphatase secreted into the medium during growth of Tetrahymena pryiformis strain W was purified about 900-fold by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified acid phosphatase was homogenous as judged by polycrylamide gel electrophoresis and was found to be a glycoprotein. Its carbohydrate content was about 10% of the total protein content. The native enzyme has a molecular weight of 120 000 as determined by gel filtration and 61 000 as determined by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The acid phosphatase thus appears to consist of two subunits of equal size. The amino acid analysis revealed a relatively high content of asparic acid, glutamic acid and leucine. The purified acid phosphatase from Tetrahymena had a rather broad substrate specificity; it hydrolyzed organic phosphates, nucleotide phosphates and hexose phosphates, but had no diesterase activity. The K_m values determined with p-nitrophenyl phosphate, adenosine 5′-phosphate and glucose 6-phosphate were 3.1·10^?4 M, 3.9·10^?4 M and 1.6·10^?3 M, respectively. The optima pH for hydrolysis of three substrates were similar (pH 4.6). Hg²⁺ and Fe³⁺ at 5 mM were inhibitory for the purified acid phosphatase, and fluoride, L-(+)-tartaric acid and molybdate also inhibited its cavity at low concentrations. The enzyme was competitively inhibited by NaF (K_i=5.6·10^?4 M) and by L-(+)-tartaric acid (K_i = 8.5·10^?5 M), while it was inhibited noncompetitively by molybdate K_i = 5.0·10^?6 M). The extracellular acid phosphatase purified from Tetrahymena was indistinguishable from the intracellular enzyme in optimum pH, K_m, thermal stability and inhibition by NaF.     

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35 000 both before and after 4 M urea treatment. Its activity was specific for the β-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A_0.5 = 0.6 μM). This enzyme had a molecular weight of approx. 70 000 which was reduced to approx. 35 000 after treatment with 4 M urea. It dephosphorylated both the α- and β-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

THE EFFECTS OF PHOSPHITE ON PHOSPHATE STARVATION RESPONSES OF ULVA LACTUCA (ULVALES,CHLOROPHYTA)1

Effects of phosphite (Phi) on phosphate (Pi) starvation responses were determined in Ulva lactuca L. by incubation in Pi‐limited (1 μM NaH₂PO₄) or Pi‐sufficient (100 μM NaH₂PO₄) seawater containing 0–3 mM Phi. Exposure to 1 μM NaH₂PO₄ decreased the growth rate and the content of free Pi and esterified‐P but increased the activities of extracellular alkaline phosphatase (EC 3.1.2.1) and intracellular acid phosphatase (ACP; EC 3.1.2.2); two ACP isozymes observed by activity staining on isoelectric focussing (IEF) gel were induced. The K_m value of Pi uptake rate was decreased by incubation with 1 μM NaH₂PO₄ and the decrease in K_m value was inhibited by 2 mM Phi, reflecting the operation of a high‐affinity Pi uptake system at low Pi concentrations. In the presence of Phi, the growth rate of Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. As Phi concentrations were increased from 0 to 2 mM, the free Pi contents in both Pi‐sufficient and Pi‐starved thalli decreased, but the esterified‐P contents in Pi‐starved thalli increased, whereas those in Pi‐sufficient thalli increased at 1 mM Phi and decreased at 2 mM Phi. Cell wall localized AP activity in both Pi‐sufficient and Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM. Intracellular ACP activity in Pi‐starved thalli decreased as Phi concentrations were increased from 0 to 2 mM but was not affected in Pi‐sufficient thalli. The induction of ACP isozyme activity and high‐affinity Pi uptake system in Pi‐starved thalli was inhibited by Phi. The present investigation shows that Phi interrupts the sensing mechanisms of U. lactuca to Pi‐limiting conditions.     

Characterization of glucoamylase from Lactobacillus amylovorus ATCC 33621

Summary An intracellular glucoamylase, purified from Lactobacillus amylovorus, reacted selectively with polysaccharides. Kinetic studies indicated low affinity for maltose and maltotriose (K_m 58 g/ml and 178 g/ml) and higher affinity for starch and dextrin (K_m 0.01 g/ml and 0.02 g/ml). Glucoamylase was inhibited almost 50% by 10 mM glucose. Cu²⁺ and Pb²⁺ inhibited glucoamylase at 1.0 mM but EDTA and other metal chelators had no effect on the enzyme activity. Acarbose and Tris inhibited the enzyme by 84% and 98%, respectively at 1 mM, while iodoacetate and p-chloromecuribenzoic acid inhibited activity by 98% and 78%, respectively at 10 mM. The purified enzyme was thermolabile at temperatures greater than 55°C and thus has potential for application in the brewing industry.