scholarly journals ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

2006 ◽  
Vol 400 (3) ◽  
pp. 477-484 ◽  
Author(s):  
Nick Sirijovski ◽  
Ulf Olsson ◽  
Joakim Lundqvist ◽  
Salam Al-Karadaghi ◽  
Robert D. Willows ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Molecular Mass ◽  
Mass Fraction ◽  
Biosynthetic Pathway ◽  
Rhodobacter Capsulatus ◽  
Gel Filtration ◽  
Protoporphyrin Ix ◽  
High Molecular Mass ◽  
Magnesium Chelatase ◽  
Ring Complex

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.

scholarly journals Effect of diosgenin on biliary cholesterol transport in the rat

1993 ◽  
Vol 291 (3) ◽  
pp. 793-798 ◽  
Author(s):  
A Thewles ◽  
R A Parslow ◽  
R Coleman
Keyword(s):  
Bile Salt ◽  
Molecular Mass ◽  
Bile Salts ◽  
Mass Fraction ◽  
Gel Permeation Chromatography ◽  
High Molecular Mass ◽  
Cholesterol Transport ◽  
Biliary Cholesterol ◽  
Lower Molecular Mass ◽  
Salt Depletion

Biliary cholesterol output in rats was stimulated over 3-fold by feeding diosgenin for 5 days, whereas biliary outputs of phospholipid and bile salts were not changed by diosgenin feeding. Isolating and perfusing the liver without bile salts resulted in a rapid and substantial decrease in biliary bile salt output; bile salt depletion abolished the diosgenin-induced increment in biliary cholesterol output, showing that the diosgenin-elevated biliary cholesterol output was bile-salt-dependent. Diosgenin treatment also produced a significant decrease in biliary alkaline phosphodiesterase I. Fresh bile obtained from control and diosgenin-fed rats was subjected to gel-permeation chromatography in order to separate different-sized biliary cholesterol carriers. Two major peaks of cholesterol were eluted, with cholesterol also being eluted between the peaks. The cholesterol peak eluted at the lower molecular mass (20-30 kDa) was observed in all bile samples. The higher-molecular-mass peak, which was eluted at the void volume, was not observed in all biles; control biles contained very little high-molecular-mass form of cholesterol, whereas biles from the diosgenin group contained up to 47% of cholesterol in the high-molecular-mass fraction. Diosgenin treatment produced a range of elevated biliary cholesterol values which positively correlated with the proportion of cholesterol contained in the high-molecular-mass fraction (r = 0.98). The results show that diosgenin induced a marked bile-salt-dependent increase in biliary cholesterol output and a shift in biliary cholesterol transport to higher-molecular-mass structures.

ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact

2008 ◽  
Vol 410 (3) ◽  
pp. 631-631 ◽  
Author(s):  
N. Sirijovski ◽  
U. Olsson ◽  
J. Lundqvist ◽  
S. Al-Karadaghi ◽  
R. D. Willows ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Biosynthetic Pathway ◽  
Magnesium Chelatase

Purification and characterization of fructans with β‐2, 1‐ and β‐2, 6‐glycosidic linkages suitable for enzyme studies

2020 ◽  
Author(s):  
GD BONNETT ◽  
Ian Sims ◽  
JA ST. JOHN ◽  
RJ SIMPSON
Keyword(s):  
Molecular Mass ◽  
Small Proportion ◽  
Gel Filtration ◽  
Triticum Aestivum L ◽  
High Molecular Mass ◽  
Enzyme Assays ◽  
Purification And Characterization ◽  
Linear Molecules ◽  
Low Levels

Fructan pentasaccharides were purified, in quantities suitable for use as substrates for enzyme assays, from Neosugar‐p‐(Meijj Seika Kaisha Ltd. Japan), tubers of Helianthus tuberosus L., L., and stems and leaf sheaths of Triticum aestivum L by a combination of gel‐filtration and RP‐HPLC. Fructan of higher molecular mass (mean DP = 30) was purified from Leaves of Lolium rigidum Gaud, that had been induced to accumulate fructan and characterized along; with the commercially available fructan from Cichorium intybus L. (Sigma, St Louis, USA) (mean DP = 33). The fructan pentasaccharide purified from H. tuberosus was found to contain exclusively 2, 1‐linked fructose and terminal fructose and terminal glucose, and was identified as (1, 1, 1)‐kestopentatise. The fructan pentasaccharide purified from Neosugar‐P also contained (1,1,1)‐kestopentaose. although the presence of fructan Klinked glucose and 1 % 2, 6‐linked fructose indicated that a small proportion of other kestopentaoses were present, The fructan pentasaccharide purified from T aestivum consisted of almost exclusively 2,6‐linked fructose and terminal glucose and terminal fructose and was considered to contain predominantly (6,6,6)‐kestopentaose. The presence of 1 % 2,1,6)‐linked fructose indicated the sample also contained a small proportion of branched kestopentanse. The high molecular mass fructan from C. intybus was found to comprise linear molecules containing only 2,1‐linked fructose, terminal glucose and terminal fructose‐ High molecular mass fructan from L. rigidum contained predominantly 2. h‐linked fructose, had predominantly internal glucose, indicated by 2 %, 1.6‐linked glucose, low levels of branching, indicated 2 % 2,1,6‐linked fructose residues; and 1% of the residues were 2,1 ‐linked fructose. Copyright © 1994, Wiley Blackwell. All rights reserved

1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis

2017 ◽  
Vol 474 (12) ◽  
pp. 2095-2105 ◽  
Author(s):  
Artur Sawicki ◽  
Shuaixiang Zhou ◽  
Kathrin Kwiatkowski ◽  
Meizhong Luo ◽  
Robert D. Willows
Keyword(s):  
Rhodobacter Capsulatus ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Magnesium Chelatase ◽  
Orthologous Protein ◽  
Substrate Complex ◽  
Motor Complex ◽  
Histidine Phosphorylation ◽  
Mg Chelatase ◽  
Stimulation Of

Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme ‘motor’ complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-histidine within ChlD. Proteomic analysis indicates that phosphorylation occurs at a conserved His residue in the C-terminal integrin I domain of ChlD. Comparative analysis of the ChlD phosphorylation with enzyme activities of various ChlI/ChlD complexes correlates the phosphorylation by ChlI2 with stimulation of Mg-chelatase activity. Mutation of the H641 of CrChlD to E641 prevents both phosphorylation and stimulation of Mg-chelatase activity, confirming that phosphorylation at H641 stimulates Mg-chelatase. The properties of ChlI2 compared with ChlI1 of Chlamydomonas and with ChlI of Oryza, shows that ChlI2 has a regulatory role in Chlamydomonas.

scholarly journals Magnesium chelatase from Rhodobacter sphaeroides: initial characterization of the enzyme using purified subunits and evidence for a BchI–BchD complex

1999 ◽  
Vol 337 (2) ◽  
pp. 243-251 ◽  
Author(s):  
Lucien C. D. GIBSON ◽  
Poul Erik JENSEN ◽  
C. Neil HUNTER
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Chlorophyll Biosynthesis ◽  
Gel Filtration ◽  
Protoporphyrin Ix ◽  
Photosynthetic Bacterium ◽  
Affinity Tags ◽  
Magnesium Chelatase ◽  
Highly Active ◽  
Mg Chelatase

The enzyme magnesium-protoporphyrin IX chelatase (Mg chelatase) catalyses the insertion of Mg into protoporphyrin IX, the first committed step in (bacterio)chlorophyll biosynthesis. In the photosynthetic bacterium Rhodobacter sphaeroides, this reaction is catalysed by the products of the bchI, bchDand bchH genes. These genes have been expressed in Escherichia coli so that the BchI, BchD and BchH proteins are produced with N-terminal His6 affinity tags, which has led to the production of large amounts of highly purified, highly active Mg chelatase subunits from a single chromatography step. Furthermore, BchD has been purifed free of contamination with the chaperone GroEL, which had proven to be a problem in the past. BchD, present largely as an insoluble protein in E. coli, was purified in 6 M urea and refolded by addition of BchI, MgCl2 and ATP, yielding highly active protein. BchI/BchD mixtures prepared in this way were used in conjunction with BchH to determine the kinetic parameters of R. sphaeroides Mg chelatase for its natural substrates. We have been able to demonstrate for the first time that BchI and BchD form a complex, and that Mg2+ and ATP are required to establish and maintain this complex. Gel filtration data suggest that BchI and BchD form a complex of molecular mass 200 kDa in the presence of Mg2+ and ATP. Our data suggest that, in vivo, BchD is only folded correctly and maintained in its correct conformation in the presence of BchI, Mg2+ and ATP.

Purification and characterization of fructans with β‐2, 1‐ and β‐2, 6‐glycosidic linkages suitable for enzyme studies

2020 ◽  
Author(s):  
GD BONNETT ◽  
Ian Sims ◽  
JA ST. JOHN ◽  
RJ SIMPSON
Keyword(s):  
Molecular Mass ◽  
Small Proportion ◽  
Gel Filtration ◽  
Triticum Aestivum L ◽  
High Molecular Mass ◽  
Enzyme Assays ◽  
Purification And Characterization ◽  
Linear Molecules ◽  
Low Levels

Fructan pentasaccharides were purified, in quantities suitable for use as substrates for enzyme assays, from Neosugar‐p‐(Meijj Seika Kaisha Ltd. Japan), tubers of Helianthus tuberosus L., L., and stems and leaf sheaths of Triticum aestivum L by a combination of gel‐filtration and RP‐HPLC. Fructan of higher molecular mass (mean DP = 30) was purified from Leaves of Lolium rigidum Gaud, that had been induced to accumulate fructan and characterized along; with the commercially available fructan from Cichorium intybus L. (Sigma, St Louis, USA) (mean DP = 33). The fructan pentasaccharide purified from H. tuberosus was found to contain exclusively 2, 1‐linked fructose and terminal fructose and terminal glucose, and was identified as (1, 1, 1)‐kestopentatise. The fructan pentasaccharide purified from Neosugar‐P also contained (1,1,1)‐kestopentaose. although the presence of fructan Klinked glucose and 1 % 2, 6‐linked fructose indicated that a small proportion of other kestopentaoses were present, The fructan pentasaccharide purified from T aestivum consisted of almost exclusively 2,6‐linked fructose and terminal glucose and terminal fructose and was considered to contain predominantly (6,6,6)‐kestopentaose. The presence of 1 % 2,1,6)‐linked fructose indicated the sample also contained a small proportion of branched kestopentanse. The high molecular mass fructan from C. intybus was found to comprise linear molecules containing only 2,1‐linked fructose, terminal glucose and terminal fructose‐ High molecular mass fructan from L. rigidum contained predominantly 2. h‐linked fructose, had predominantly internal glucose, indicated by 2 %, 1.6‐linked glucose, low levels of branching, indicated 2 % 2,1,6‐linked fructose residues; and 1% of the residues were 2,1 ‐linked fructose. Copyright © 1994, Wiley Blackwell. All rights reserved

scholarly journals Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation

2000 ◽  
Vol 352 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Poul E. JENSEN ◽  
James D. REID ◽  
C. Neil HUNTER
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Chlorophyll Biosynthesis ◽  
Protoporphyrin Ix ◽  
Cysteine Residue ◽  
Enzyme Inactivation ◽  
Cysteine Residues ◽  
Magnesium Chelatase ◽  
Synechocystis Pcc6803 ◽  
Chelation Reaction

The enzyme magnesium protoporphyrin chelatase catalyses the insertion of magnesium into protoporphyrin, the first committed step in chlorophyll biosynthesis. Magnesium chelatase from the cyanobacterium Synechocystis PCC6803 has been reconstituted in a highly active state as a result of purifying the constituent proteins from strains of Escherichia coli that overproduce the ChlH, ChlI and ChlD subunits. These individual subunits were analysed for their sensitivity to N-ethylmaleimide (NEM), in order to assess the roles that cysteine residues play in the partial reactions that comprise the catalytic cycle of Mg2+ chelatase, such as the ATPase activity of ChlI, and the formation of ChlI–ChlD–MgATP and ChlH–protoporphyrin complexes. It was shown that NEM binds to ChlI and inhibits the ATPase activity of this subunit, and that prior incubation with MgATP affords protection against inhibition. Quantitative analysis of the effects of NEM binding on ChlI-catalysed ATPase activity showed that three out of four thiols per ChlI molecule are available to react with NEM, but only one cysteine residue per ChlI subunit is essential for ATPase activity. In contrast, the cysteines in ChlD are not essential for Mg2+ chelatase activity, and the formation of the ChlI–ChlD–ATP complex can proceed with NEM-treated ChlI. Neither the ATPase activity of ChlI nor NEM-modifiable cysteines are therefore required to form the ChlI–ChlD–MgATP complex. However, this complex cannot catalyse magnesium chelation in the presence of the ChlH subunit, protoporphyrin and Mg2+ ions. The simplest explanation for this is that in an intact Mg2+ chelatase complex the ATPase activity of ChlI drives the chelation process. NEM binds to ChlH and inhibits the chelation reaction, and this effect can be partially alleviated by pre-incubating ChlH with magnesium and ATP. We conclude that cysteine residues play an important role in the chelation reaction, in respect of the ChlI–MgATP association, ATP hydrolysis and in the interaction of ChlH with MgATP and protoporphyrin IX.

Properties of High-Molecular-Mass Placental Alkaline Phosphatases in Normal Pregnancy Sera

1998 ◽  
Vol 35 (4) ◽  
pp. 515-521 ◽  
Author(s):  
Makoto Matsushita ◽  
Tsutomu Irino ◽  
Masakazu Minowa ◽  
Tsugikazu Komoda ◽  
Torgny Stigbrand
Keyword(s):  
Alkaline Phosphatase ◽  
Pregnant Women ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Molecular Mass ◽  
Placental Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Serum Samples ◽  
Triton X

We examined the appearance of high-molecular-mass placental alkaline phosphatases (HPLAPs) in the serum of normal pregnant women by means of polyacrylamide gel electrophoresis (PAGE) in the presence of Triton X-100. The HPLAPs were undetectable or only slightly detectable by PAGE in the absence of Triton X-100. The HPLAPs were detected in all sera sampled during the last trimester of pregnancy. The catalytic activities of total placental alkaline phosphatase (TPLAP) and HPLAPs were correlated (r = 0.96) and the ratio of HPLAPs/TPLAP catalytic activity was 0.20 (0.06) (mean and SD) in 40 serum samples from pregnant women. The HPLAPs appear to be formed from a common dimeric placental alkaline phosphatase (PLAP) (common-PLAP), as judged by the fact that they were formed again after removal of HPLAPs from serum by gel filtration. The formation of HPLAPs was more prominently observed with the faster fractions of gel filtration. The apparent molecular mass of the HPLAPs in pregnancy serum was 720 K Da by gel filtration. HPLAPs were not converted to common-PLAP by phosphatidylinositol-specific phospholipase (PIPL) C and PIPL-D treatments. The HPLAPs were selectively incorporated into liposomes consisting of phosphatidylcholine/cholesterol, and most of the PIPL-D-treated PLAP could form HPLAPs, while a small amount of PLAP could not form HPLAPs. On the other hand, HPLAPs in pregnant women's sera and HPLAPs prepared from partially purified PLAP in vitro could be converted to common-PLAP by brief treatment with subtilisin. However, the highly purified PLAP could not form HPLAPs in the presence of Triton X-100. These results suggest that PIPL-D-resistant and PLAP-associated serum protein may regulate the conversion of PLAP to HPLAP in the presence of Triton X-100.

The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle

1999 ◽  
Vol 112 (21) ◽  
pp. 3703-3712 ◽  
Author(s):  
Z. Lygerou ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Molecular Mass ◽  
Fission Yeast ◽  
Origin Recognition Complex ◽  
Gel Filtration ◽  
S Phase ◽  
High Molecular Mass ◽  
Homologous Proteins ◽  
Origin Recognition

The origin recognition complex (ORC) binds to the well defined origins of DNA replication in budding yeast. Homologous proteins in other eukaryotes have been identified but are less well characterised. We report here the characterisation of a fission yeast ORC complex (SpORC). Database searches identified a fission yeast Orc5 homologue. SpOrc5 is essential for cell viability and its deletion phenotype is identical to that of two previously identified ORC subunit homologues, SpOrc1 (Orp1/Cdc30) and SpOrc2 (Orp2). Co-immunoprecipitation experiments demonstrate that SpOrc1 forms a complex with SpOrc2 and SpOrc5 and gel filtration chromatography shows that SpOrc1 and SpOrc5 fractionate as high molecular mass complexes. SpORC subunits localise to the nucleus in a punctate distribution which persists throughout interphase and mitosis. We developed a chromatin isolation protocol and show that SpOrc1, 2 and 5 are associated with chromatin at all phases of the cell cycle. While the levels, nuclear localisation and chromatin association of SpORC remain constant through the cell cycle, one of its subunits, SpOrc2, is differentially modified. We show that SpOrc2 is a phosphoprotein which is hypermodified in mitosis and is rapidly converted to a faster migrating isoform as cells proceed into G(1) in preparation for S-phase.

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