Phosphatases and phosphodiesterases isolated from the red king crab (Paralithodes camtschatica) hepatopancreas

Five enzymes have been isolated from the hepatopancreas of the red king crab Paralithodes camtschatica by means of ion exchange and gel chromatography: two acid (AcP) and one alkaline (AlkP) phosphomonoesterases, one alkaline phosphodiesterase (AlkPI), and one acid phosphodiesterase (AcPD). The pH optimum values of these enzymes are: AlkPs and AlkPD, 7.5; AcP, 5.5; and AcPD, 5.0. The activity of AlkP and AlkPD demands Mg²⁺ ions. The molecular weights of the enzymes (kDa) are the following: AlkP, 80; AcPs, 80 and 82; AlkPD, 51; and AcPD, 57. The enzymes are relatively thermostable (ID 50 from 47 to 62°C). AlkP is inhibited by NaCl (IC 50 at 0.4 M). The AcP, AcPD, and AlkPD activities are tolerant of high ionic strength.     

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.     

Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Rat liver lowM r phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.     

Protective potentials of a potentized homeopathic drug, Lycopodium-30, in ameliorating azo dye induced hepatocarcinogenesis in mice

The protective potentials of a potentized homeopathic drug, Lycopodium-30, prepared from extract of spores of a plant, Lyocopodium clavatum (Fam: Lycopodiaceae) and used as a remedy for various liver ailments, have been tested in mice chronically fed p-dimethyl amino azo benzene (p-DAB) – an initiator, and phenobarbital (PB) – a promoter of hepatic cancer, by using some cytogenetic endpoints like chromosome aberrations (CA), micronuclei (MN), mitotic index (MI) and sperm head abnormality (SHA), and toxicity biomarkers like acid and alkaline phosphatases (AcP and AlkP, respectively), alanine and aspartate amino transferases (ALT and AST, respectively) and lipid peroxidation (LPO) and reduced glutathione (GSH) activities. The effects of chronic treatment of the carcinogens were assessed at different intervals of fixation, namely, at day 7, 15, 30, 60, 90 and day 120, and compared with that of mice fed conjointly with the carcinogens and the homeopathic remedy. Both the assay systems indicated considerable protective potentials of the homeopathic remedy against p-DAB induced hepatocarcinogenesis in mice.     

Cytochemical study of cells of primary and disseminated ascite Yoshida tumor cells

The different distribution of cytochemically demonstrable enzymes: lactate dehydrogenase (LDH, 1.1.1.27), succinate dehydrogenase (SDH, 1.3.99.1), dihydrofolate reductase (DHFR, 1.5.1.3), acid phosphatase (AcP, 3.1.3.2) and alkaline phosphatase (ALP, 3.1.3.1), has been documented in Yoshida ascites hepatoma cells in vivo or stored at 80 degrees C. The dehydrogenase activities (LDH, SDH, DHFR) show a strong reaction in all samples. An increased level of these enzyme activities has been observed in the malignant cells spreading through the organs of tumor bearing rats. On the contrary, in the same samples, acid and alkaline phosphatase activities are very low. The strong dehydrogenase activities observed in Yoshida ascite cells stress the rapid turnover of tumor cells. Our results indicate that the histochemical method may be a useful tool to detect the scattered tumor cells. Furthermore, the cytochemical methods allow the characterization of the metabolic pathways employed by the primary and disseminated tumor cells.     

Acid and alkaline phosphatases of Capnocytophaga species. I. Production and cytological localization of the enzymes

The two hydrolytic enzymes, acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase, of the three types species of Capnocytophaga were examined. Both enzymes were produced constitutively, with their activity highest in C. ochracea strain 25. These two degradative enzymes (approximately 10% of the total activity) were released into the growth medium during the latter stages of growth, both as soluble and membrane-bound enzymes. When grown in the presence of high concentrations of organic phosphates, the synthesis and expression of AcP and AlP was unaltered. Cyto- and immuno-chemical localization situated the phosphatases in the periplasmic space, at the cell surface, and in membranous vesicles.     

5'-Nucleotide phosphodiesterase and alkaline phosphatase in tumor cells: evidence for existence of novel species in the cytosol

Characteristics of 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) and alkaline phosphatase (EC 3.1.3.1) activities in tumor cell lines of human and murine origin were examined. Of the 15 cell lines tested, 5'-nucleotide phosphodiesterase activity in 13 cell lines and alkaline phosphatase activity in 10 cell lines were inhibited by N-ethylmaleimide and activated by dithiothreitol (N-ethylmaleimide-sensitive), and suggested to be SH-enzymes. In contrast, the two phosphohydrolases from normal tissues were inactivated by dithiothreitol, but not by N-ethylmaleimide (dithiothreitol-sensitive). There was only one tumor cell line in which both activities were dithiothreitol-sensitive. Human hepatoma PLC/PRF/5 cells appear to possess both types of 5'-nucleotide phosphodiesterase and alkaline phosphatase, and the subcellular distribution of these enzymes in this cell line was investigated. Dithiothreitol-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase of PLC/PRF/5 cells were localized in the plasma membrane as in normal tissues, but N-ethylmaleimide-sensitive phosphohydrolases were soluble cytosolic proteins. N-Ethylmaleimide-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase activities from other cell lines were also recovered in the cytosol. Molecular masses of cytosolic N-ethylmaleimide-sensitive phosphohydrolases were apparently smaller than their membrane-bound dithiothreitol-sensitive counterparts, as judged from gel filtration. It was concluded that many tumor cell lines lack plasma membrane 5'-nucleotide phosphodiesterase and alkaline phosphatase, but express enzymes with similar activities in the cytosol, with properties clearly distinguishable from enzymes so far characterized.     

Properties of a series of tegumental membrane-bound phosphohydrolase activities of Schistosoma mansoni.

1. Incubation of Schistosoma mansoni for 5 min in a phosphate-buffered medium, pH 7.4, released tegumental material containing the following phosphohydrolase activities: alkaline phosphatase, 5'-nucleotidase, glycerol-2-phosphatase, glucose 6-phosphatase, phosphodiesterase and ATPase. 2. Maximum activity of these enzymes was measured at pH 9.5; however, the phosphodiesterase and ATPase activities were also appreciable at pH 7.0. 3. Solubilization of the released tegumental material in 1% Triton X-100 followed by gel filtration distinguished three peaks of enzyme activity: an ATPase (mol.wt. greater than 1000 000), a phosphodiesterase (mol.wt. 1 000 000) and an alkaline phosphomonoesterase with broad specificity (mol.wt. 232 000). 4. The ATPase activity was highly activated by 10 mM-Mg2+ or 1 mM-Ca2+ and was inhibited by chelating agents. Ouabain, Na+ and K+ had little effect on enzyme activity, whereas activity was increased by 50% in the presence of calmodulin. The phosphodiesterase activity was highest in the presence of 100 mM-Na+ or -K+, and 10 mM-Mg2+ or -Ca2+. Alkaline phosphatase activity was also stimulated by 100 mM-Na+ or -K+, and 10 mM-Mg2+; however Ca2+ inhibited at greater than 1 mM. 5. Surface iodination of parasites followed by detergent solubilization and gel filtration of the released tegumental membranes indicated that these enzymes were not accessible. A major surface component, apparent mol.wt. 80 000, was iodinated. 6. Rabbit anti-(mouse liver 5'-nucleotidase) antibodies did not inhibit the phosphohydrolase activities. However, an immunoglobulin G fraction from sera of mice chronically infected with S. mansoni partially inhibited alkaline phosphatase activity, but was without effect on the phosphodiesterase and ATPase activities. 7. The location of the enzymes in the double membrane of the tegument and their significance in host-parasite interactions is discussed.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

Crystal structure and structural stability of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3

To elucidate the structural basis for the high stability of acylphosphatase (AcP) from Pyrococcus horikoshii OT3, we determined its crystal structure at 1.72 A resolution. P. horikoshii AcP possesses high stability despite its approximately 30% sequence identity with eukaryotic enzymes that have moderate thermostability. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic counterparts. The crystal structure of P. horikoshii AcP shows the same fold betaalphabetabetaalphabeta topology and the conserved putative catalytic residues as observed in eukaryotic enzymes. Comparison with the crystal structure of bovine common-type AcP and that of D. melanogaster AcP (AcPDro2) as representative of eukaryotic AcP revealed some significant characteristics in P. horikoshii AcP that likely play important roles in structural stability: (1) shortening of the flexible N-terminal region and long loop; (2) an increased number of ion pairs on the protein surface; (3) stabilization of the loop structure by hydrogen bonds. In P. horikoshii AcP, two ion pair networks were observed one located in the loop structure positioned near the C-terminus, and other on the beta-sheet. The importance of ion pairs for structural stability was confirmed by site-directed mutation and denaturation induced by guanidium chloride.     

Sensitivity of lysosomal enzymes to the plant alkaloid sanguinarine: comparison with other SH-specific agents

The influence of the benzo[c]phenanthridine alkaloid sanguinarine on some lysosomal enzyme activities was investigated. Sanguinarine inhibits lysosomal hydrolases in homogenates of cultured mouse fibroblasts. After incubation of mouse fibroblasts in culture with 100 microM sanguinarine an approximately 50% decrease in the activities of N-acetyl-beta,D-glucosaminidase (NAGA), beta-galactosidase (GAL), arylsulfatase and acid lipase was observed. Because the biological activity of sanguinarine might arise from the interaction of its iminium cation with enzyme thiol groups, we compared its effect on NAGA, GAL and acid phosphatase (AcP) activities with the effects of SH-specific reagents p-chloromercuribenzoic acid (CPMA) and N-ethylmaleimide (NEM). Treatment of lysosomal fractions with millimolar concentrations of sanguinarine induces a dose-dependent inhibition of the enzymes; for example, 0.6 mM sanguinarine causes approximately a 40% decrease in AcP and NAGA activities. NEM has similar effects, and increasing the preincubation temperature from 0 degrees C to 37 degrees C intensifies the inhibition due to both agents. CPMA also inhibits the activity of GAL (IC50 0.7 microM), AcP (IC50 12.5 microM) and NAGA (IC50 6.8 microM) in a dose-dependent manner but is more potent than sanguinarine or NEM. Comparative analysis of the primary structures of these enzymes using the program BLAST reveals the presence of highly conserved cysteine residues, which confirms the importance of thiol-groups for their activities. Thus, both the experimental observations obtained in this study and the literature data imply a significant role of redox-based mechanisms in regulating lysosomal functional activity.     

Acetyl Phosphate as a Primordial Energy Currency at the Origin of Life

Metabolism is primed through the formation of thioesters via acetyl CoA and the phosphorylation of substrates by ATP. Prebiotic equivalents such as methyl thioacetate and acetyl phosphate have been proposed to catalyse analogous reactions at the origin of life, but their propensity to hydrolyse challenges this view. Here we show that acetyl phosphate (AcP) can be synthesised in water within minutes from thioacetate (but not methyl thioacetate) under ambient conditions. AcP is stable over hours, depending on temperature, pH and cation content, giving it an ideal poise between stability and reactivity. We show that AcP can phosphorylate nucleotide precursors such as ribose to ribose-5-phosphate and adenosine to adenosine monophosphate, at modest (~2%) yield in water, and at a range of pH. AcP can also phosphorylate ADP to ATP in water over several hours at 50 °C. But AcP did not promote polymerization of either glycine or AMP. The amino group of glycine was preferentially acetylated by AcP, especially at alkaline pH, hindering the formation of polypeptides. AMP formed small stacks of up to 7 monomers, but these did not polymerise in the presence of AcP in aqueous solution. We conclude that AcP can phosphorylate biologically meaningful substrates in a manner analogous to ATP, promoting the origins of metabolism, but is unlikely to have driven polymerization of macromolecules such as polypeptides or RNA in free solution. This is consistent with the idea that a period of monomer (cofactor) catalysis preceded the emergence of polymeric enzymes or ribozymes at the origin of life.     

Reactivation of inhibited bone acid phosphatase and its significance in bone histomorphometry

Despite biochemical demonstration of acid phosphatase (AcP) activation or reactivation in bone, few attempts have been made to show similar effects histochemically. Bones from growing rats, when fixed in 4% buffered formaldehyde at room temperature and demineralized in 5% formic acid, exhibited expected inactivation of AcP. The inhibited AcP, however, was reactivated by pre-incubation of sections for 1 hr at 37 degrees C in the following buffers: 0.2 M Tris, 0.2 M glycine, 0.2 M NaHCO3, or 0.1 M borax, as well as in alkaline water, but not in 0.2 M Na2HPO4 (all at pH 9). The reactivation was (a) site-specific (e.g., osteoclasts, osteoblasts, osteocytes, and cement lines), (b) temperature- and pH-dependent, (c) unaffected by OH- or SH--binding agents or by an alkaline phosphatase inhibitor, and (d) inhibited completely by 10 mM Na2HPO4. The reactivation process, much simplified and/or more effective than with the methods previously reported, was observed in all 83 human biopsy bones embedded in methyl methacrylate and in human bones stored in cold buffered formaldehyde for 7 months. This study demonstrates a unique method for reactivating and thus localizing the inhibited AcP in bones, and suggests possible applications in bone histomorphometry.     

Characterization of phosphohydrolase activity in bovine spleen extracts: identification of a bis(p-nitrophenyl)phosphate-hydrolyzing activity (phosphodiesterase IV) which also acts on adenosine triphosphate

Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p-nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl)-and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4-methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate).     

Enzyme activity and acute phase proteins in milk utilized as indicators of acute clinical E. coli LPS-induced mastitis

The importance of non-visual and on-line monitoring of udder health increases as the contact between humans and animals decreases, for example, in robotic milking systems. Several indicator systems have been introduced commercially, and a number of techniques are currently in use. This study describes the kinetics of seven indigenous milk parameters for monitoring udder inflammation in an Escherichia coli lipopolysaccharide (LPS, endotoxin)-induced mastitis model. Proportional milk from LPS-infused quarters was compared with milk from parallel quarters, which were placebo-treated with sterile 0.9% NaCl solution. Somatic cell counts (SCCs), the acute phase proteins (APP), that is, milk amyloid A (MAA) and haptoglobin (Hp), and the enzymes N-acetyl-β-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), alkaline phosphatase (AP) and acid phosphatase (AcP) were measured at fixed intervals during the period from -2 to +5 days after LPS and NaCl infusions. All parameters responded significantly faster and were more pronounced to the LPS infusions compared with the NaCl infusions. All parameters were elevated in the proportional milk collected at the first milking 7 h after infusion and developed a monophasic response, except Hp and MAA that developed biphasic response. SCC, LDH, NAGase and Hp peaked at 21 h followed by AP, AcP and MAA peaking at 31 h with the highest fold changes seen for MAA (23 780×), LDH (126×), NAGase (50×) and Hp (16×). In the recovery phase, AP, AcP and Hp reached base levels first, at 117 h, whereas LDH, NAGase and MAA remained elevated following the pattern of SCC. Minor increases of the milk parameters were also seen in the neighboring (healthy) quarters. Distinction between inflamed and healthy quarters was possible for all the parameters, but only for a limited time frame for AP and AcP. Hence, when tested in an LPS mastitis model, the enzymes LDH, NAGase and AP in several aspects performed equally with SCC and APP as inflammatory milk indicators of mastitis. Furthermore, these enzymes appear potent in the assessment of a valuable time sequence of inflammation, a necessary ingredient in modeling of programs in in-line surveillance systems.     

Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

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

Effects of bile salts on the plasma membranes of isolated rat hepatocytes.

The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20--30% of the plasma-membrane enzymes 5'-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10--20% of the 5'-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5--2.0mM) than either glycocholate or taurocholate (12--16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly 'soluble' form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.     

Biochemical and Cytochemical Evidence for the Polar Concentration of Periplasmic Enzymes in a “Minicell” Strain of Escherichia coli

A number of "surface" enzymes of Escherichia coli (i.e., among those selectively released by osmotic shock) all displayed higher specific activities in extracts of minicells than in extracts of typical rod forms; these enzymes included alkaline phosphatase, cyclic phosphodiesterase, acid hexose monophosphatase, 5'-nucleotidase, and ribonuclease I. In addition, alkaline phosphatase, cyclic phosphodiesterase, and acid hexose monophosphatase were cytochemically localized to regions of minicell periplasm that resembled reactive polar enlargements of the periplasm in rod forms. In contrast, a number of "internal" cytoplasmic enzymes (inorganic pyrophosphatase, beta-galactosidase, glutamine synthetase, polynucleotide phosphorylase, and ribonuclease II) showed elevated or similar specific activities in extracts of rod forms versus extracts of minicells. A specific heat-labile inhibitor for 5'-nucleotidase, known to occur in the cytoplasm, also showed no enrichment in minicells. These findings indicate that the "surface" enzymes are segregated in vivo into the terminal minicell buds, possibly because these enzymes are concentrated in the polar enlargements of the periplasm in typical rod forms.