scholarly journals Alkaline Phosphatase Mutants of Bacillus subtilis

1975 ◽  
Vol 28 (3) ◽  
pp. 323 ◽  
Author(s):  
A RGlenn
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Control System ◽  
Phosphate Starvation ◽  
Wild Type ◽  
Alkaline Phosphatases ◽  
Wild Type Enzyme ◽  
Vegetative Cells ◽  
Specific Alkaline Phosphatase ◽  
Low Levels

Alkaline phosphatases from vegetative and sporulating cells of B. subtilis have been shown previously to be identical in all criteria examined. Despite this, 15 mutants producing low levels of the phosphatase during phosphate starvation of vegetative cells have been shown to produce high levels of the sporulation-specific alkaline phosphatase. It has been shown by imrnunochemical means that seven of these mutants when starved of phosphate produce low levels of normal wild-type enzyme. The sporulation form of the enzyme from one mutant (P-l00) has been shown to be identical with the phosphatases from vegetative and sporulating cells of the wild type. It is proposed that all the mutants have regulatory defects in the control of the alkaline phosphatase from vegetative cells but nevertheless retain an intact structural gene for the enzyme and the control system for the phosphatase during sporulation.

Retention at the cis-Golgi and delayed degradation of tissue-non-specific alkaline phosphatase with an Asn153→Asp substitution, a cause of perinatal hypophosphatasia

2002 ◽  
Vol 361 (3) ◽  
pp. 473-480 ◽  
Author(s):  
Masahiro ITO ◽  
Norio AMIZUKA ◽  
Hidehiro OZAWA ◽  
Kimimitsu ODA
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Control Process ◽  
Brefeldin A ◽  
Dependent Manner ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Specific Alkaline Phosphatase ◽  
Cell Surface Biotinylation ◽  
Quality Control Process

Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn153→Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308–314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse—chase experiments showed that newly synthesized TNSALP (N153D) remained endo-β-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface.

scholarly journals REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: IDENTIFICATION OF THE STRUCTURAL GENE FOR REPRESSIBLE ALKALINE PHOSPHATASE

Genetics ◽  
1976 ◽  
Vol 84 (2) ◽  
pp. 175-182
Author(s):  
John F Lehman ◽  
Robert L Metzenberg
Keyword(s):  
Alkaline Phosphatase ◽  
Linkage Group ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Type A ◽  
Phosphate Metabolism ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Group V ◽  
Low Levels

ABSTRACT Five additional mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase. The mutations in these strains map at a previously assigned locus on Linkage Group V designated pho-2 (Gleason and Metzenberg 1974). The five new mutants, as well as three previously isolated by Gleason and Metzenberg (1974), were examined for the presence of cross-reacting material to antibody prepared against purified wild-type enzyme. Two of the mutants produced high levels of cross-reacting material, thus providing evidence that the pho-2 locus includes the structural gene for the repressible alkaline phosphatase. Two revertants were obtained from one of the mutants that contained cross-reacting material. Neither revertant produced an enzyme that could be distinguished physicochemically from that of wild type. A method for measuring very low levels of repressible alkaline phosphatase in crude extracts is also described.

scholarly journals Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

1991 ◽  
Vol 279 (1) ◽  
pp. 35-41 ◽  
Author(s):  
R Chambert ◽  
M F Petit-Glatron
Keyword(s):  
Bacillus Subtilis ◽  
Intermediate Formation ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Chain Elongation ◽  
Directed Mutagenesis ◽  
Water Mixture ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

Endometrial phosphatases, β-glucuronidase and cathepsin D during menstrual cycle and pre-implantation stages of gestation in the rhesus monkey (Macaca mulatta)

1988 ◽  
Vol 118 (1) ◽  
pp. 142-146 ◽  
Author(s):  
Jayasree Sengupta ◽  
Deepa Talwar ◽  
B. C. Paria ◽  
D. Ghosh
Keyword(s):  
Alkaline Phosphatase ◽  
Menstrual Cycle ◽  
Rhesus Monkey ◽  
Cathepsin D ◽  
Acid Hydrolases ◽  
Alkaline Phosphatases ◽  
Serum Progesterone ◽  
Low Levels ◽  
Ovulatory Period ◽  
Estradiol 17Β

Abstract. β-glucuronidase, cathepsin D, acid and alkaline phosphatases were studied in rhesus monkey endometrium during the menstrual cycle (day –6 to day + 10) and pre-implantation stages (day +3 to day +6) of gestation, with day 0 considered as the day of ovulation. Acid hydrolases exhibited low levels in proliferative phase endometria followed by their gradual rise in the secretory phase of the menstrual cycle. Despite no shifts in the levels of serum progesterone and estradiol-17β, the pre-implantation period was, however, associated with distinct changes in enzyme profiles characterized by lower absolute levels (P < 0.05) of acid phosphatase and β-glucuronidase on days 3 to 6 of gestation, whereas cathepsin D activity declined significantly (P < 0.05) on days 5 and 6. Alkaline phosphatase showed a characteristic rise during the pre-ovulatory period with a gradual lowering of its level in post-ovulatory phase endometria of a non-fertile cycle; in contrast, during early gestation, alkaline phosphatase activity showed a marked elevation (P < 0.05) on days 5 and 6 of gestation. The significance of these findings is discussed.

scholarly journals New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation

1989 ◽  
Vol 260 (2) ◽  
pp. 491-497 ◽  
Author(s):  
L Hederstedt ◽  
L O Hedén
Keyword(s):  
Bacillus Subtilis ◽  
Succinate Dehydrogenase ◽  
Active Site ◽  
Amino Acid Position ◽  
Biochemical Properties ◽  
Cysteine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Succinate Oxidation ◽  
E Coli

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

scholarly journals Effects of Nonpolar Mutations in Each of the Seven Bacillus subtilis mrp Genes Suggest Complex Interactions among the Gene Products in Support of Na+ and Alkali but Not Cholate Resistance

2000 ◽  
Vol 182 (20) ◽  
pp. 5663-5670 ◽  
Author(s):  
Masahiro Ito ◽  
Arthur A. Guffanti ◽  
Wei Wang ◽  
Terry A. Krulwich
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Vesicles ◽  
Alkali Resistance ◽  
Gene Products ◽  
Wild Type ◽  
Upstream Gene ◽  
Complex Interactions ◽  
Low Levels ◽  
The Individual ◽  
Secondary Membrane

ABSTRACT The Bacillus subtilis mrp (multiple resistance and pH) operon supports Na+ and alkali resistance via an Na+/H+ antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpFmutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans. Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na+ efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na+ resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na+sensitivity at both pH 7.0 and 8.3. The mrpA andmrpD mutants were particularly sensitive to alkaline pH even without added Na+. While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships among mrp genes as well. All the mrp mutants, especially the mrpA,-B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type. Expression of an upstream gene, maeN, that encodes an Na+/malate symporter, was coordinately regulated withmrp, although it is not part of the operon.

scholarly journals Involvement of bone-specific alkaline phosphatase and procollagen I carboxy-terminal propeptide as predictors of early fracture risk in Chinese Children with juvenile osteoporosis: An interventional clinical trial

2018 ◽  
Vol 13 (2) ◽  
pp. 164
Author(s):  
Yu Wang ◽  
Jingxin Zhao ◽  
Lingwei Kong ◽  
Yu Jin
Keyword(s):  
Clinical Trial ◽  
Vitamin D ◽  
Alkaline Phosphatase ◽  
Bone Fracture ◽  
Specific Alkaline Phosphatase ◽  
Bone Specific Alkaline Phosphatase ◽  
Low Levels ◽  
Carboxy Terminal ◽  
To Receive

<p class="Abstract">This clinical trial was designed to understand whether the children with juvenile osteoporosis receiving tablet containing vitamin D and calcium had lower incidence of bone fracture compared to the children receiving a diet rich in calcium, vitamin D, and protein. We assessed whether plasma levels of bone-specific alkaline phosphatase (BSAP) and procollagen I carboxy-terminal propeptide levels (PIPC) could be used as predictors of early bone fracture in children. A total of 120 children of either gender with a juvenile osteoporosis were enrolled and randomized (1:1 ratio) to receive tablet containing vitamin D and calcium (n=60) or diet rich in calcium, vitamin D, and protein (n=60), and undergone follow-up for up to 3 years.  Blood sample was collected and BSAP and PIPC levels were measured. The results suggested that therapeutic intervention (vitamin D and calcium) does not predict bone fracture in children. However, correlations analysis revealed that the decreased level of BSAP and PIPC were associated with higher incidence of fracture. The results suggest that the low levels of BSAP and PIPC cause increase susceptibility of fracture among children with juvenile osteoporosis.</p>

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