A locus regulating N-acetylglucosaminidase synthesis during development in dictyostelium

The cellular specific activity of N-acetylglucosaminidase increases during development in Dictyostelium discoideum. A monoclonal antibody which specifically recognizes Mr 68,000 and 67,000 forms of N-acetylglucosaminidase was used to show that changes in the relative rate of enzyme synthesis during development parallel the pattern of enzyme accumulation. Developmental and regulatory mutants were isolated to study the relationship between development and enzyme accumulation. No evidence was obtained for any dependence of enzyme accumulation on those genes that are required for aggregation. However, a separate regulatory locus was identified which is involved in enzyme accumulation. Mutations in this gene, nagC, prevent enzyme accumulation during development by preventing an increase in the relative synthetic rate of N-acetylglucosaminidase. The accumulation of other enzymes is unaffected and the mutation causes no developmental defects other than those caused by the loss of N-acetylglucosaminidase activity. The nagC mutation, which is recessive, maps to linkage group VI and is therefore unlinked to the structural gene for N-acetylglucosaminidase.     

The Structural Gene for α-Mannosidase-1 in DICTYOSTELIUM DISCOIDEUM

We have isolated 4 independent mutations affecting alpha-mannosidase-1, a developmentally regulated activity in Dictyrostelium discoideum. Three of these result in a thermolabile alpha-mannosidase-1 activity. One mutation also affects the substrate affinity (Km) of the activity. In diploids these mutations show a gene dosage effect and are all alleles. The structural gene for alpha-mannosidase-1, as defined by these mutations, defines a new linkage group, linkage group VI. alpha-mammosidase 1 is probably a homopolymer with subunits of 54,000 daltons. We have also mapped two temperature-sensitive-for-growth mutations onto two previously defined linkage groups.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Identification of the Structural Gene for Repressible Acid Phosphatase

A mutant of Neurospora crassa with an altered repressible acid phosphatase has been isolated. The enzyme is much more thermolabile than that of wild type, and has an increased Michaelis constant. Tests of allelic interactions (in partial diploids) and in vitro mixing experiments were consistent with the mutation being in the structural gene for the enzyme. This gene, pho-3, was found to be located in the right arm of Linkage Group IV (LGIV). Thus, pho-3 and the structural gene for repressible alkaline phosphatase, pho-2 (LG V), map in separate linkage groups and cannot be part of the same operon. Neither of these structural genes is linked to the known regulatory genes, nuc-1 (LG I), nuc-2 (LG II), and preg (LG II).     

Alkaline phosphatase in the lactating bovine mammary gland and the milk fat globule membrane. Release by phosphatidylinositol-specific phospholipase C.

1. Alkaline phosphatase is covalently bound to bovine mammary microsomal membranes and milk fat globule membranes through linkage to phosphatidylinositol as demonstrated by the release of alkaline phosphatase following treatment with phosphatidylinositol-specific phospholipase C. 2. The release of alkaline phosphatase from the pellet to the supernatant was demonstrated by enzyme assays and electrophoresis. 3. Electrophoresis of the solubilized enzymes showed that the alkaline phosphatase of the microsomal membranes contained several isozymes, while only one band with alkaline phosphatase activity was seen in the fat globule membrane. 4. Levamisole and homoarginine were potent inhibitors of the alkaline phosphatase activities in both membrane preparations and in bovine liver alkaline phosphatase, but not in calf intestinal alkaline phosphatase.     

The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum

A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.     

Modulation of alkaline phosphatase activity in alveolar type II like cells

Summary Alveolar type II like cells (ALT II) represent a small subpopulation of alveolar type II cells, which is able to proliferate, can be passaged and possess many characteristics of differentiated adult type II cells. A correlation was found between the growth and development of ALT II cells in culture and their alkaline phosphatase activity. Unlike alveolar type II cells, which lose the activity in culture, ALT II cells regain the activity and maintain it for a long culture period. Quantitative histochemical analysis of the stained cells indicate that 80% of the cells at days 15–20 in culture are alkaline phosphatase positive. Inhibition studies indicate that alkaline phosphatase from ALT II cells and freshly isolated type II cells were similar. The inhibition of ALT II alkaline phosphatase byl-levamisole and its heat stability are similar to that of the bone enzyme and differ from the intestinal enzyme. Alkaline phosphatase expression is considered part of the differentiated phenotype of these cells. Therefore, the presence of this enzyme in ALT II cells adds support to the notion that these cells maintain many aspects of mature alveolar type II cells.     

Alterations in Activity of Phosphatases during the Peridinium Bloom in Lake Kinneret

Acid and alkaline phosphatases have been isolated from Peridinium cinctum f. westii (Dinophyceae) during an algal bloom in Lake Kinneret. Acid phosphatase activity was fairly constant over the entire period of the bloom, although fluctuations in activity appeared to correlate with the chlorophyll content of the cells. Histochemical studies showed that the enzyme was localized inside the cell. Alkaline phosphatase activity was very low until May, a month after the peak of the bloom, when it increased sharply. Polyacrylamide gel electrophoresis revealed one or two bands of alkaline phosphatase that increased in intensity as the bloom progressed. However, the highest activity of the enzyme (in the last sample collected) corresponded to a new, very intense band on the gels. Similarly to acid phosphatase, alkaline phosphatase was also localized inside the cell. The appearance of alkaline phosphatase is probably related to the available phosphate concentration in the lake, although the influence of other factors that may contribute to the induction of the enzyme cannot be ruled out.     

Modulation of alkaline phosphatase activity in alveolar type II like cells

Alveolar type II like cells (ALT II) represent a small subpopulation of alveolar type II cells, which is able to proliferate, can be passaged and possess many characteristics of differentiated adult type II cells. A correlation was found between the growth and development of ALT II cells in culture and their alkaline phosphatase activity. Unlike alveolar type II cells, which lose the activity in culture, ALT II cells regain the activity and maintain it for a long culture period. Quantitative histochemical analysis of the stained cells indicate that 80% of the cells at days 15-20 in culture are alkaline phosphatase positive. Inhibition studies indicate that alkaline phosphatase from ALT II cells and freshly isolated type II cells were similar. The inhibition of ALT II alkaline phosphatase by L-levamisole and its heat stability are similar to that of the bone enzyme and differ from the intestinal enzyme. Alkaline phosphatase expression is considered part of the differentiated phenotype of these cells. Therefore, the presence of this enzyme in ALT II cells adds support to the notion that these cells maintain many aspects of mature alveolar type II cells.     

Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proehancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picornole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D -glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.     

Measurement of serum alkaline phosphatase

Alkaline phosphatase is a commonly requested enzyme test in clinical chemistry. However, the enzyme is not particularly substrate specific, which has led to a proliferation of methods for its analysis. It can exhibit a variable instability effect depending on the techniques required for its storage or analysis. Methods can also be highly dependent on sample isoenzyme distribution and reagent purity, leading to problems in the quality control of its analysis and in the comparison of results obtained from different methods. Alkaline phosphatase is not tissue specific and this may on occasion lead to uncertainty in the interpretation of its measured activity in blood serum. In recent years there has been a number of attempts to standardize methodology for this and other enzymes. Perhaps an alternative approach to the measurement of alkaline phosphatase activity will alleviate some of the problems encountered.     

Alkaline and acid phosphatases from the extensively halotolerant bacteriumHalomonas elongata

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

Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene.

Escherichia coli alkaline phosphatase constitutive mutants carrying a pst or a phoS mutation and a plasmid-bearing gene phoA+ excreted into the growth medium up to 50% of the total alkaline phosphatase production. This excretion was pH dependent and did not involve drastic modifications of the cell envelope. Alkaline phosphatase accounted for 80% of total released proteins. Amplification of gene phoA+ was a necessary condition for excretion to occur. When the beta-lactamase structural gene bla+ was coamplified with gene phoA+, both enzymes were excreted. pst-transformed excretory strains did not show the pleiotrophic phenotype previously described for lky mutants.     

Thermolabile alkaline phosphatase from Sphingobacterium antarcticus a psychrotrophic bacterium from Antarctica

Eight psychrotrophic strains belonging to four different genera were screened for the presence of cold-active alkaline phosphatase in sonicated cell homogenates. An approximately 1000-fold higher activity than E. coli was detected in two psychrotrophic strains of Sphingobacterium antarcticus and one mesophilic strain of Flavobacterium multivorum. The enzymes from the psychrotrophs showed maximum activity at 37°C and were also found to be active at 0°C. Alkaline phosphatase from one psychrotrophic Sphingobacterium lost 97% of its activity when it was heated for 10 min at 62°C. This enzyme was partially purified and characterised. The production of the enzyme was repressed when the organism was grown in the presence of phosphates and its activity was inhibited on preincubation with inorganic phosphates and ethylene diamine tetracetic acid. Potassium permanganate and potassium periodate did not inhibit the activity of the enzyme. The biotechnological importance of the enzyme is discussed.     

Recessive Lethal Mutations and the Maintenance of Duplication-Bearing Strains of Dictyostelium discoideum

Recessive lethal mutations have been isolated and used to maintain n + 1 aneuploid strains of Dictyostelium discoideum carrying a duplication of part or all of linkage group VII. The recessive lethal mutations, relA351 and relB352, arose spontaneously in diploids; no mutagenic treatment was used in the isolation of these mutations. The probable gene order on linkage group VII is: centromere, relB couA, bsgB, cobA, relA. Maintenance of aneuploids disomic for linkage group VII was made possible by complementation of a rel mutation on each linkage group VII homologue by the corresponding wild-type allele on the other linkage group VII homologue. The duplication-bearing disomic strains were slow-growing and produced faster-growing sectors on the colony edge. Haploid sectors probably arise by a combination of mitotic recombination and subsequent loss of one homologue, diploid sectors may be formed by chromosome doubling to 2n + 2, followed by chromosome loss to return to 2n, and aneuploid sectors may arise by deletion or new mutation.     

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55–60 kDa, as determined by SDS–PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.     

Hematologic and enzymatic analysis of Ctenopharyngodon idella x Hypophthalmichthys nobilis F1 hybrids

1. Erythrocyte counts, hemoglobin concentrations and hematocrit values were determined for diploid and triploid Ctenopharyngodon idella X Hypophthalmichthys nobilis hybrids and the parental species. 2. Comparisons of diploid and triploid hybrids with the parental species revealed low erythrocyte counts for triploids, high mean corpuscular hemoglobin values for triploids, elevated hematocrits for diploids and triploids and similar hemoglobin concentrations for all fish. 3. Alkaline phosphatase, aldolase, and lactate dehydrogenase specific activities were determined spectrophotometrically. Levels of specific activity of these enzymes in the hybrids were consistently elevated above that of the parental species. These higher levels of enzyme activities in hybrids were probably the result of a breakdown in gene regulation.     

Solubility properties of alkaline phosphatase from matrix vesicles

Alkaline phosphatase has been extracted from matrix vesicles of a calcifying cartilage with 0.15 M KCl, 0.4 M guanidinium chloride and 0.05 M deoxycholate/50% butanol mixture. The catalytic properties of the three extracts have been compared. Although the highest amount of enzyme activity is extracted with the latter reagent (55%), some of it is also extracted with KCl (11%) and guanidinium (7%). By submitting isolated matrix vesicles to a short time sonication the distribution pattern of the alkaline phosphatase activity in the extracts is clearly modified, as the amount extracted with KCl increases from 14 to 50% and the portion extracted with deoxycholate decreases from 55 to 27% of the total enzyme activity of matrix vesicles. The enzymatic preparations were comparable on the basis of specific activities, affinity for the substrates (p-nitrophenylphosphate, ATP), thermostability, sensitivity to inhibitors and activators. By electrofocusing a value of pI = 4.15 was found for the alkaline phosphatase of matrix vesicles independently of the extraction medium. These results contradict the concept that alkaline phosphatase is exclusively an intrinsic membrane protein.     

Alkaline phosphatase fusions: sensors of subcellular location.

Alkaline phosphatase fusions allow genes to be identified solely on the basis of their protein products being exported from the cytoplasm. Thus, the use of such fusions helps render biological processes which involve cell envelope and secreted proteins accessible to a sophisticated genetic analysis. Furthermore, alkaline phosphatase fusions can be used to locate export signals. Specifying such signals is an important component of studies on the structure of individual cell envelope proteins. The basis of the alkaline phosphatase fusion approach is the finding that the activity of the enzyme responds differently to different environments. Thus, the activity of the fusion protein gives evidence as to its location. This general approach of using sensor proteins which vary in their function, depending on their environment, could be extended to the study of other sorts of problems. It may be that certain enzymes will provide an assay for localization to a particular subcellular compartment, if the environment of the compartment differs from that of others. For instance, the lysosome is more acidic than other intracellular organelles. A gene fusion system employing a reporter enzyme that could show activity only at the pH of the lysosome could allow the detection of signals determining lysosomal localization. Analogous types of enzymes may be used as probes for other subcellular compartments.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.