Two forms of O-methyltransferase in tobacco cell suspension culture

An S-adenosyl-l-methionine: o-dihydric phenol O-methyltransferase was isolated from tobacco cell suspension culture and was partially purified by (NH₄)₂SO₄ precipitation and successive chromatography on DEAE-Sepharose, Sephacryl S-200 and hydroxyapatite columns. It catalysed the O-methylation of 3 cinnamic acids, two coumarins and two flavonoids, but to different extents. Results obtained from polyacrylamide gel electrophoresis, m-/p-methylation ratios and mixed substrate experiments indicated the existence of two forms of the enzyme which were resolved by chromatography on DEAE-cellulose. One form (MW 74000, pI 6.1, opt. pH 7.3) catalysed the meta-methylation of caffeic acid, while the other (MW 70000, pI 6.3, opt. pH 8.3) mediated the para-methylation of quercetin, though each form exhibited some activity against other substrates.     

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.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

Liquid-liquid phase separation of a monoclonal antibody and nonmonotonic influence of Hofmeister anions

Liquid-liquid phase separation was studied for a monoclonal antibody in the monovalent salt solutions of KF, KCl, and KSCN under different pH conditions. A modified Carnahan-Starling hard-sphere model was utilized to fit the experimental data, establish the liquid-liquid coexistence curve, and determine antibody-antibody interactions in the form of T_c (critical temperature) under the different solution conditions. The liquid-liquid phase separation revealed the complex relationships between antibody-antibody interactions and different solution conditions, such as pH, ionic strength, and the type of anion. At pH 7.1, close to the pI of the antibody, a decrease of T_c versus ionic strength was observed at low salt conditions, suggesting that the protein-protein interactions became less attractive. At a pH value below the pI of the antibody, a nonmonotonic relationship of T_c versus ionic strength was apparent: initially as the ionic strength increased, protein-protein interactions became more attractive with the effectiveness of the anions following the inverse Hofmeister series; then the interactions became less attractive following the direct Hofmeister series. This nonmonotonic relationship may be explained by combining the charge neutralization by the anions, perhaps with the ion-correlation force for polarizable anions, and their preferential interactions with the antibody.     

Regulation of extracellular acid phosphatase biosynthesis by culture conditions in entomopathogenic fungusMetarhizium anisopliae strain CQMa102

Metarhizium anisopliae is an imperfect entomopathogenic fungus. Once invading into its host,M. anisopliae needs to absorb basic nutrients such as phosphorus from the host haemolymph. A large number of phosphorylated compounds in haemolymph cannot be directly utilised by the fungal cell and must be hydrolysed into available form by phosphatase before ingested. Aims of this paper were to investigate optimum fermentation conditions for production of acid phosphatase and phosphatase isoenzymes byMetarhizium anisopliae. The optimum fermentation conditions were: glucose, 20 g/l; (NH₄)₂SO₄, 2 g/l; casein, 4 g/l; MgSO₄, 0.5 g; KCl, 0.5 g; microelement salt solution, 10 ml; inoculum size, 1×10⁷ spores per 100 ml medium; initial medium pH, 6.0. Under these conditions, the highest total acid phosphatase activity was 3.05 U/ml in 4 days at 27 °C and 160 rpm. Synthesis of the acid phosphatase was repressed by 0.01% inorganic phosphate in culture medium. The spectrum of isoenzymes produced byM. anisopliae varied depending on the phosphorus source employed in the culture. A specific isoform with pI 9.45 was induced by casein, and another isoform of pI 8.21 was induced by phytic acid and disodium phenyl phosphate.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50 kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5–7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K_m = 2.1 × 10⁻³ M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase.The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Salt-stimulated Adenosine Triphosphatase from Smooth Microsomes of Turnip

The turnip (Brassica rapa L.) microsome fraction contains both a Mg²⁺-inhibited acid phosphatase and a salt-stimulated Mg²⁺-activated ATPase. However, as the pH optimum of the ATPase was 8.0 to 8.5, the acid phosphatase activity could be eliminated by assaying at or above pH 7.8. The ATPase was concentrated in a fraction equivalent to the smooth microsomal membranes and was not due to fragments of mitochondria. The salt-stimulated activity showed specificity for anions rather than cations. The activity was further stimulated by carbonyl cyanide m-chloro-phenylhydrazone (CCCP), 2,4-dinitrophenol, valinomycin, nigericin, and NH₄Cl. There was a synergistic effect between CCCP and valinomycin. Activity was insensitive to oligomycin phlorizin, ouabain, and atractylate. Based on similarity to the chloroplast ATPase, it was proposed that this ATPase was situated on the outside of the vesicle.     

Isolation and characterization of a tartrate-sensitive splenic acid phosphatase in Gaucher's disease

The acid phosphatase activity that is increased in the spleens of patients with Gaucher's disease can be separated into two principal isoenzymes by chromatography on sulphopropyl-Sephadex. The acid phosphatase species that is resistant to inhibition by l-(+)-tartrate is retained by the cation-exchange resin while the tartrate-sensitive species passes through. We have isolated and characterized the tartrate-sensitive acid phosphatase (designated SP_I) from the spleen of a patient with the adult (type 1) form of Gaucher's disease. SP_I acid phosphatase, representing approximately 30 to 50% of the total acid phosphatase activity in a detergent (Triton X-100) extract of spleen tissue, has been purified approximately 400-fold to a specific activity of 48 units/mg of protein (substrate, 4-methylumbelliferyl phosphate). The final preparation of acid phosphatase contains at least two protein components—each with phosphatase activity—when analyzed by polyacrylamide gel electrophoresis at pH 8.9 or isoelectric focusing. SP_I acid phosphatase exhibits a broad substrate specificity and catalyzes the hydrolysis of a variety of artificial and natural phosphate-containing compounds including p-nitrophenyl phosphate, α-naphthyl phosphate, phosphoenolpyruvate, and CMP. The enzyme is inhibited by l-(+)-tartrate, sodium fluoride, and ammonium molybdate and has the following properties: pH optimum, 4.5; K_m on 4-methylumbelliferyl phosphate, 44 μm; pI, 3.8–4.1; M_r, 177,400; s_20,w, 6.8.     

Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. The Ca²⁺-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [³²P]-phosphatidylinositol from pH5.0 to pH8.5. However, the activity in the range pH7.0–8.5 could not be recovered after precipitation with (NH₄)₂SO₄; most of the enzyme activity was recovered in the 30–50% fraction and showed a single sharp pH optimum at 5.5. 2. The cytosolic supernatant was analysed by isoelectric focusing on acrylamide gels, and assay at pH5.5. Four peaks of phosphodiesterase activity were found at pI ranges 7.4–7.2, 6.0–5.8, 4.8–4.4 and 4.2–3.8. 3. The cytosolic supernatant was also applied to a chromatofocusing column, and again assayed at pH5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a second peak at pH6.0–5.85; a third broad peak with a wide range pH5.3–4.2; and a fourth peak, which was eluted by washing the column with 1m-NaCl, suggesting an isoenzyme with a pI below 4.0 (supported by the result of the isoelectric focusing). 4. If all the chromatofocusing fractions were assayed at pH7.0 or 8.0 (at 1mm-Ca²⁺), only a single sharp peak was detected, with a pI of 4.6–4.8. This peak disappeared on (NH₄)₂SO₄ fractionation (30–50%) of the cytosolic supernatant, whereas the four peaks with activity at pH5.5 were virtually unaffected. 5. The four activities (assayed at pH5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. 6. We conclude that the Ca²⁺-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity, and its isoelectric properties.     

Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H₂O₂ was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type lb Cu(II).     

Study of effects of antibody to protein S100 on ionic channels of input and output currents of identified neurons of the snail Helix lucorum

In the course of electrophysiological experiments, two types of the neurons of the edible snail Helix lucorum were detected, which responded by different way to application of antibodies to the neuron-specific calcium-binding S 100 protein (AS1000). Under effect of AS100, frequency of the action potential (AP) generation in the spontaneously active V1, V3, V17, and RPa6 cells decreased, whereas in V4 and V6 cells increased. On addition of quinine solution the AP generation frequency of these neurons decreased more than twice, while the AP duration (t _S) rose 6 times. The combined action of AS100 and quinine did not change statistically significantly the AP generation frequency, membrane potential (MP) and AP generation threshold (AP_t), as compared with the effect of AS100 in saline. The value of the AP duration (t _S) increased 1.6 times, which was less pronounced as compared with the quinine action in saline. This means that AS100 prevents an increase of the AP duration after the quinine application (block of the Ca-depended K-channels). The main AS100 effect at the level of the ionic currents is shown to consist in a decrease of the maximal value of the input current, on average, by 20%, while of the output current, on average, by 12%.     

Multiple forms of acidic endopeptidase from germinated barley

An endopeptidase preparation from germinated barley Hordeum vulgare L., cv. Trophy, purified by affinity chromatography and density-gradient electrofocusing, consisted of three or four components. The preparation was only partly resolved by electrofocusing, with evidence of three possible components (pI 4.15, 4.28, and 4.37). Gel filtration on Sephadex G-75 yielded an asymmetrical peak, the major part of which corresponded to a molecular weight of 14,100, with evidence of one larger and two smaller components. The activity of the preparation was sulfhydryl-dependent; cysteine was the most effective of several sulfhydryl compounds tested. The preparation was sensitive to O₂ in the absence of metal chelating agents and was inhibited by sulfhydryl reagents. It showed very narrow concentration tolerances for both cysteine and a substrate, N,N-dimethylhemoglobin. The Km value on N,N-dimethylhemoglobin at pH 3.8 was 0.064 to 0.067% (w/v) substrate; V_max was 0.80 to 0.83 A₃₄₀ per hour. Normal enzyme activity and molecular-size distribution were observed when the endopeptidases were extracted in the inhibited state and subsequently reactivated, thus ruling out the possibility that the enzymes might be autolytic artifacts that arose during extraction and purification.     

X-ray crystal structure of Vibrio alkaline phosphatase with the non-competitive inhibitor cyclohexylamine

BackgroundPara-nitrophenyl phosphate, the common substrate for alkaline phosphatase (AP), is available as a cyclohexylamine salt. Here, we report that cyclohexylamine is a non-competitive inhibitor of APs.MethodsCyclohexylamine inhibited four different APs. Co-crystallization with the cold-active Vibrio AP (VAP) was performed and the structure solved.ResultsInhibition of VAP fitted a non-competitive kinetic model (K_m unchanged, V_max reduced) with IC₅₀ 45.3 mM at the pH optimum 9.8, not sensitive to 0.5 M NaCl, and IC₅₀ 27.9 mM at pH 8.0, where the addition of 0.5 M NaCl altered the inhibition to the level observed at pH 9.8. APs from E. coli and calf intestines were less sensitive to cyclohexylamine, whereas an Antarctic bacterial AP was similar to VAP in this respect. X-ray crystallography at 2.3 Å showed two binding sites, one in the active site channel and another at the surface close to dimer interface. Antarctic bacterial AP and VAP have Trp274 in common in their active-sites, that takes part in binding cyclohexylamine. VAP variants W274A, W274K, and W274H gave IC₅₀ values of 179 mM, 188 mM and 187 mM, respectively, at pH 9.8.ConclusionsThe binding of cyclohexylamine in locations at the dimeric interface and/or in the active site of APs may delay product release or reduce the rate of catalytic step(s) involving conformational changes and intersubunit communications.General significanceCyclohexylamine is a common chemical in industries and used as a counterion in substrates for alkaline phosphatase, a clinically important and common enzyme in the biosphere.     

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.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Purification and Characterization of Phosphoglycolate Phosphatase from the Cyanobacterium Coccochloris peniocystis

The properties and role of the enzyme phosphoglycolate phosphatase in the cyanobacterium Coccochloris peniocystis have been investigated. Phosphoglycolate phosphatase was purified 92-fold and had a native molecular mass of approximately 56 kilodaltons. The enzyme demonstrated a broad pH optimum of pH 5.0 to 7.5 and showed a relatively low apparent affinity for substrate (K_m = 222 micromolar) when compared to that from higher plants. The enzyme required both an anion and divalent cation for activity. Mn²⁺ and Mg²⁺ were effective divalent cations while Cl⁻ was the most effective anion tested. The enzyme was specific for phosphoglycolate and did not show any activity toward a variety of organic phosphate esters. Growth of the cells on high CO₂ and transfer to air did not result in any significant change in phosphoglycolate phosphatase activity. Competitive inhibition of C. peniocystis triose phosphate isomerase by phosphoglycolate was demonstrated (K_i = 12.9 micromolar). These results indicate the presence of a specific noninducible phosphoglycolate phosphatase whose sole function may be to hydrolyze phosphoglycolate and prevent phosphoglycolate inhibition of triose phosphate isomerase.     

The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO₂ hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K_m of 2.8 mm. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate (K_i = 1.22 × 10^?3M), arsenate (K_i = 1.17 × 10^?3M), and molybdate (K_i = 1.34 × 10^?7M), with these inhibitors having no effect on the CO₂ hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO₂ hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO₂ hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.     

Peptidases of yeast and filamentous forms ofCandida albicans

A study of the peptidases of the yeast and filamentous forms ofCandida albicans H-317 was carried out. At least three distinct peptidase activities in cell extracts of the yeast and filamentous phases were separated by gel filtration column chromatography or native polyacrylamide gel electrophoresis and measured by an enzyme-coupled colorimetric assay. The first peak of hydrolyzing activity to elute from a Sephadex G-200 column was designated as peak I, an aminopeptidase(s) with broad substrate specificity, and was characterized as follows: It had a molecular weight of 550,000, pH optima 7.5 to 8.0, andK_m of 1 × 10⁻⁴M with trimethionine as substrate, and it was inhibited by EDTA. The activity of peak I showed stereospecificity since it hydrolyzedd-methionyl-l-methionyl-l-methionine andl-methionyl-l-methionyl-d-methionine but notl-methionyl-d-methionyl-l-methionine. The second peak to elute from the gel filtration column was designated peak II. Its molecular weight was estimated to be 120,000, it had a pH optimum of 7.0, and it was inhibited by EDTA. TheK_m with trimethionine as substrate for the yeast and filamentous phase peak II was 3.2 × 10⁻⁴ and 7.5 × 10⁻⁵M, respectively. In addition to peak I and peak II peptidase(s), a dipeptidase activity with a molecular weight of 150,000 was detected in both the yeast and filamentous phases. Thus, a multiplicity of peptidases was present in cell extracts ofC. albicans and no major differences were noted in the partially purified peptidases from either the yeast or filamentous phase.     

Induction of acid phosphatase in cotton seedlings: Enzyme purification,subunit structure and kinetic properties

About a 16-fold rise in acid phosphatase (EC 3.1.3.2) activity was observed during the early stages of germination of cotton embryos. Administration of cyclobeximide to the germinating embryos significantly blocked the enhancement of acid phosphatase activity. This indicated that translational activity was essential for the induction of enzyme activity. Conclusive proof for the de novo synthesis of the enzyme was obtained by showing the incorporation of ³⁵S from ³⁵SO²⁻₄ into the cysteine residues of the purified acid phosphatase. The enzyme was purified (1046-fold) to electrophoretic homogeneity by ammonium sulphate fractionation, CM-Sephadex C-50 and affinity chromatography on concanavalin A-Agarose. PAGE gave two isozyme bands. The M, of the phosphatase was 200 k as determined by molecular sieving on Sephadex G-200. SDS-PAGE of acid phosphatase revealed a single band of M 55 k. Thus the native enzyme is a tetramer of four identical subunits. The K_m of the enzyme with p-nitrophenyl phosphate was 0.5 mM. Optimal enzyme activity was observed at pH 5.0, using p-nitrophenyl phosphate as substrate. The enzyme activity remained linear for 105 min at 37° and was proportional to the concentration of protein within the range 0.6–2.4 μg.