p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O₂-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.     

Biochemical Changes Associated With Brassica juncea Seed Development. IV. Acid and Alkaline Phosphatases

Changes in acid and alkaline phosphatase activities in cytoplasmic and wall-bound fractions of developing mustard (Brassica juncea) seed were studied. Growth was measured by seed dry weight and water content. Seed dry weight data were fitted to a cubic polynomial equation. Seed water content and dry matter accumulation was significantly correlated. Cytoplasmic acid and alkaline phosphatase activities were substantially less in the cytoplasmic fraction than the wall-bound fraction. Wall-bound acid phosphatase activity was low initially, but high levels were maintained after day 25, indicating a relationship with dry matter accumulation. The results suggest that acid phosphatase plays an important role during mustard seed development. Received February 19, 1998; accepted May 6, 1999     

Isolation and characterization of a high molecular weight stable pink form of uteroferrin from uterine secretions and allantoic fluid of pigs

A pink, high molecular weight form of uteroferrin (Uf) has been isolated from uterine secretions and allantoic fluid of pigs. This protein fraction (denoted FIII) which is relatively stable under physiological conditions of pH, ionic strength, and temperature has a molecular weight of about 80,000, a value approximately twice that of purple Uf (Mr approximately 35,000) isolated from a separate fraction (FIV) by gel filtration. The visible absorption spectrum, EPR signal, and acid phosphatase activity of Uf in FIII are almost identical to those of FIV Uf after the latter has been reduced by 2-mercaptoethanol. However, unlike reduced FIV Uf, the pink, high molecular form does not revert to purple, nor does it show loss of EPR signal and phosphatase activity in the presence of oxygen. In addition, it does not become purple at orthophosphate concentrations which inhibit Uf acid phosphatase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has shown that FIII consists of approximately equal amounts of Uf polypeptides (Mr = 35,000 and 37,000) and a group of three polypeptides (Mr = 40,000, 46,000, and 50,000) antigenically unrelated to Uf. The latter share a common epitope not found on Uf and are probably differentially processed forms of the same protein. FIII can be dissociated by pH conditions below 5.0, by exposure to antibodies raised against Uf or the associated polypeptides, and by sodium dodecyl sulfate at 100 degrees C. The polypeptides in FIII are not therefore linked by disulfide bonds. Treatment with dimethyl suberimidate, however, results in a cross-linked complex (Mr approximately 82,000) consisting of Uf and the associated polypeptides. It is concluded that this high Mr form of Uf is a heterodimer of fully activated Uf and a second polypeptide of unknown function.     

Atrial natriuretic factor: atrial conversion of high to low molecular weight forms

The atrial natriuretic factor elutes by gel filtration in high and low molecular weight fractions. Extraction and elution of rat atria in 1.0 M acetic acid yielded a predominance of the high molecular weight form(s); whereas when these procedures were carried out in 0.1 M acetic acid, there was a predominance of the low molecular weight forms. When partially purified high molecular weight natriuretic activity was eluted in 0.1 M acetic acid, the high molecular weight form(s) remained intact. When partially purified high molecular weight natriuretic activity was mixed with crude atrial extract in 0.1 M acetic acid, there was an apparent conversion to the low molecular weight forms. Extraction of rat atria in boiling 0.1 M acetic acid blocked this conversion. It is concluded that rat atria contain a heat labile factor that converts high molecular weight natriuretic activity to the low molecular weight forms.     

The effect of greening of sorghum leaves on the molecular weight of a complex containing 4-hydroxycinnamic Acid hydroxylase activity

During the greening of leaves of Sorghum bicolor var. Wheatland milo, the activity of 4-hydroxycinnamic (p-coumaric) acid hydroxylase in pH 6 buffered extracts was shifted from a relatively low to a high molecular weight fraction. Differences between these forms found in etiolated and green leaves were based on differential centrifugation, ammonium sulfate precipitation, and on elution patterns from Agarose A-15m. Both molecular weight forms were precipitated by protamine sulfate at pH 6, and approximately 40 to 80% of the activity of each form was associated with a 500 to 37,000g pellet when tissues were ground at pH 8 in media of either high or low osmotic concentration. Although no fraction with hydroxylase activity was ever found without any chlorogenic acid oxidase activity, the two activities frequently varied independently, and could be partially separated from each other, using the above techniques. Comparisons were made with the very small molecular weight form of 4-hydroxycinnamic acid hydroxylase characteristic of tissues of first internodes. The significance of these results in terms of possible multienzyme complexes capable of converting phenylalanine and tyrosine to cinnamic acid derivatives is discussed.     

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms ofacid phosphatase and ribonuclease from potato tubers were studiedby seasonal assays and Sephadex gel filtration. Ribonucleaseand p-nitrophenyl phosphatase activity fluctuated during storageof tubers, while ß-glycerophosphatase declined toa low level coincident with initiation of sprout growth. Inrecently-lifted tubers acid phosphatase activity occurred ina single high molecular weight peak. Two new forms of lowermolecular weight appeared during ageing of stored tubers. Theinfluence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica,on these seasonal changes was variable. No consistent effectson total hydrolase activities were observed, while post-infectionalchanges in molecular forms included a pronounced shift in themajor acid phosphatase peak. The possible significance of thismolecular weight change in infected samples is discussed inthe light of recent evidence concerning the sub-unit structureof acid phosphatase from potato tubers.     

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.     

Chloramphenicol stimulates acid phosphatase activity in germinating cotton (Gossypium hirsutum) embryos.

Low dosages of chloramphenicol (25-50 micrograms/ml) brought about a 2-4-fold stimulation of acid phosphatase activity in 48 h-germinated cotton (Gossypium hirsutum) embryos. However, at high concentrations of chloramphenicol (100-1000 micrograms/ml), there was a progressive decline in enzyme activity. The stimulatory effect of the drug on acid phosphatase activity was relatively specific, since no significant stimulation of activities of proteinase, deoxyribonuclease, ribonuclease, o-diphenolase and peroxidase was observed in germinating cotton embryos. Chloramphenicol, however, did promote the activities of isocitric lyase and alkaline phosphatase. Sephadex G-200 chromatography of the enzyme fraction revealed high (230 000)- and low (106 000)-molecular-weight multiple forms of acid phosphatase in the chloramphenicol-treated embryos, in contrast with a single molecular form (mol.wt. 106 000) in the untreated embryos. Thus the treatment of cotton embryos with chloramphenicol induced both a qualitative and a quantitative change in the acid phosphatase activity. Chloramphenicol-stimulated acid phosphatase activity was strongly inhibited when Pi was included in the germination medium. However, the control embryos showed less pronounced inhibition of enzyme activity in presence of Pi ions.     

Multiple Forms of Acid Phosphatase in Cotyledons of Vigna mungo Seedlings

Acid phosphatase from cotyledons of dark-grown Vigna mungo seedlingswas separated into four forms (la, Ib, Ha and lib) by ion-exchangercolumn chromatographies. Each form of the acid phosphatase wascharacterized for its pH dependency, substrate specificity,thermal stability, activation energy, approximate molecularweight, and the effect of metal ions and other substances onits activity. Each form of the enzyme exhibited high activitytowards ATP and ADP relative to their activity towards p-nitrophenylphosphate,but showed very low activity towards phytate, a major organicphosphate reserve in cotyledons. Among the four forms, lib wasmost distinguishable by its low molecular weight and the thermalenhancement of activity.     

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in the developing chick brain: partial characterization and levels during development.

Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase is largely expressed in chick brain tissue during development. The enzyme was purified from brain extract prepared from 19-day-old chick embryos and from adult chickens using ammonium sulfate fractionation, gel filtration on Sephadex G-75 and two DEAE-Cellulose ion-exchange chromatography steps. The purified enzymes from embryo and adult chick brains show identical molecular weight values (about 18-20 kDa) and biochemical and structural properties such as substrate specificity, sensitivity to inhibitors, and number of free reactive sulphydryl groups. These data suggest that they are the same enzyme protein. Although the total acid phosphatase activity does not change appreciably during development, the activity associated with the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase markedly increases after birth and reaches the adult values within the first week of life. Taken together, our results suggest an involvement of the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in postnatal development and maturation of chick brain tissue. The variations in tyrosine phosphorylation profile of chick brain polypeptides analyzed by Western blotting at the same developmental stages are also reported.     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

The presence of a low molecular weight acid phosphatase in liver tissue that cannot be demonstrated with the histochemical substrate naphthol AS-BI phosphate

Summary Three distinct isoenzymes of acid phosphatase have been separated from extracts of liver tissue of rats by gel filtration. These isoenzymes have molecular weights of 180,000±35,000; 74,000±11,000 and 13,000±2,500. High molecular weight isoenzymes and a low molecular weight isoenzyme of acid phosphatase (molecular weight 13,000±2,100) were also present in extracts of normal human and mouse liver tissue, and of pathologically altered liver tissue of mice in which the activity of acid phosphatase was strongly increased as a result of intraperitoneal injections of dextran solutions. Activity of acid phosphatase was determined with three substrates. The isoenzymes showed different conversion rates for the three substrates. The high molecular weight isoenzymes split the substrates 4-methylumbelliferyl phosphate, p-nitrophenyl phosphate and naphthol AS-BI phosphate. The activity was sensitive to the inhibitors fluoride and L(+)tartrate. In the pathologically altered liver tissue, which had stored dextran, the activity of these isoenzymes was strongly increased. The low molecular weight isoenzyme split 4-methylumbelliferyl phosphate and p-nitrophenyl phosphate but not naphthol AS-BI phosphate. Therefore this isoenzyme cannot be demonstrated with histochemical techniques using the substrate naphthol AS-BI phosphate. In contrast to the activity of the high molecular isoenzymes the activity of the low molecular isoenzyme was not changed in the pathologically altered liver tissue of mice and was not sensitive to the inhibitors fluoride and L(+)tartrate.This study was supported by a grant from the Prinses Beatrix Fonds, s'Gravenhage     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: II. Characterization of the Phosphate Starvation Inducible-Excreted Acid Phosphatase

Three-day-old suspension cultured cells of Lycopersicon esculentum transferred to a Pi-depleted medium had 2.7 times the excreted acid phosphatase (Apase) activity of cells transferred to a Pi-sufficient medium. Cell growth during this time period was identical for the two treatments. Excreted Apase activity was resolved into two fractions on a Sephadex G-150 column. Most of the phosphate starvation inducible (psi) enhancement in activity was in the lower molecular weight fraction. These two fractions exhibited different substrate versus pH activity profiles. With a native polyacrylamide gel electrophoresis assay, the lower molecular weight fraction resolved into two bands of activity. Both column fractions resolved into the same single band of activity with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of this enzyme was 57 kilodalton. These data indicate that L. esculentum has at least two isozymes of the psi-excreted Apase and that these isozymes may associate to form high molecular weight aggregates. Labeling studies using [³⁵S]methionine show that the psi response in tomato cells is complex and involves changes in the steady state levels of several excreted proteins.     

Acid phosphatases of Esox lucius: tissue distribution and partial characterization

The presence of different acid phosphatase classes was examined in organs and tissues of the fish Esox lucius . The enzymes were separated by Sephadex G-200 column chromatography, and characterized as regards tissue distribution, molecular weight, metal ion activation, inhibition, pH optima, temperature sensitivity, rate of hydrolysis and substrate specificity. High and low molecular weight acid phosphatases as well as high and low molecular weight Zn²⁺-dependent acid phosphatases were present in pike organs and tissues. In addition, their biochemical properties and tissue distribution are very similar to that of respective bird and mammalian enzymes.     

Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures

The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.     

Changes in phosphatase activity in phosphorus-deficient Spirodela

Summary Onset of phosphorus deficiency in Spirodela oligorrhiza was accompanied by a 50-fold increase in phosphatase activity of cell extracts. The enzyme behaved like other plant acid phosphatase, and was both inhibited and repressed by inorganic phosphate. The phosphatase activity comprised at least three isozymes. Two, of low molecular weight, were present only in P-deficient Spirodela; one of high molecular weight was also present, though in smaller amounts, in normal Spirodela. Presence of 2-thiouracil during onset of P-deficiency partly inhibited the development of phosphatase activity. The nature and role of the increased phosphatase activity in P-deficient plants are discussed.     

A STUDY OF THE COMPONENTS OF THE CORNIFIED EPITHELIUM OF HUMAN SKIN

Pulverized cornified epithelium of human skin was divided into a "soluble fraction" and a "residue." About half of the "soluble fraction" proved to be soluble epidermal keratin (keratin A); the remainder, dialyzable substances of low molecular weight. The "residue" contained epidermal keratin and resistant cell membranes of cornified cells. Epidermal keratin was found to form an oriented and dense submicroscopic structure in the cornified cells. It showed high resistance toward strong acid and moderately strong alkali solutions as well as concentrated urea. In strong alkali, reducing substances, alkaline urea, and mixtures of reducing substance with alkali, epidermal keratin dissociated and yielded a non-dialyzable derivative of high molecular weight (keratin B) which resembled true proteins. The cell membranes of cornified cells showed higher resistance toward strong alkali and reducing substance than did epidermal keratin.     

Regulation of rhodopsin dephosphorylation by arrestin

We have characterized the opsin phosphatase activities in extracts of rod outer segments and determined their relationship to known protein phosphatases. The opsin phosphatase activity in the extracts was not due to protein phosphatases 1, 2B, or 2C because it was neither stimulated by Mg2+ or Ca2+/calmodulin nor inhibited by protein phosphatase inhibitors-1 or -2. Opsin phosphatase activity in rod outer segment extracts was potently inhibited by okadaic acid (IC50 approximately 10 nM), a preferential inhibitor of protein phosphatase 2A. Moreover, during chromatography on DEAE-Sepharose, the opsin phosphatase activity co-eluted with three peaks of protein phosphatase 2A activity, termed protein phosphatases 2A0, 2A1, and 2A2. The opsin phosphatase activity of each peak was stimulated by polylysine, a known activator of protein phosphatase 2A. Finally, treatment of rod outer segment extracts with 80% ethanol at room temperature converted the activity from a high molecular weight form characteristic of the protein phosphatase 2A0, 2A1, and 2A2 species to a low molecular weight form characteristic of the protein phosphatase 2A catalytic subunit. We conclude that protein phosphatase 2A is likely to be the physiologically relevant rhodopsin phosphatase. The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin. Arrestin has no effect on the dephosphorylation of phorphorylase a, indicating that the effect was substrate-directed. It appears that dephosphorylation of the photoreceptor protein phosphorhodopsin occurs only after decay of the photoactivated protein and that this may be regulated in vivo by arrestin. The binding of arrestin to photolyzed phosphorylated rhodopsin, i.e. the binding of a regulatory protein to a protein phosphatase substrate to form a complex resistant to dephosphorylation represents a novel mechanism for the regulation of protein phosphatase 2A.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

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 50kDa 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)=2.1x10(-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.