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

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.

The ATPase Activity of the ChlI Subunit of Magnesium Chelatase and Formation of a Heptameric AAA+Ring†

Biochemistry ◽  
2003 ◽  
Vol 42 (22) ◽  
pp. 6912-6920 ◽  
Author(s):  
James D. Reid ◽  
C. Alistair Siebert ◽  
Per A. Bullough ◽  
C. Neil Hunter
Keyword(s):  
Atpase Activity ◽  
Magnesium Chelatase

ATPase activity of magnesium chelatase subunit I is required to maintain subunit D in vivo

2004 ◽  
Vol 271 (11) ◽  
pp. 2182-2188 ◽  
Author(s):  
Vanessa Lake ◽  
Ulf Olsson ◽  
Robert D. Willows ◽  
Mats Hansson
Keyword(s):  
Atpase Activity ◽  
Magnesium Chelatase

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.

scholarly journals Role of Magnesium Chelatase Activity in the Early Steps of the Tetrapyrrole Biosynthetic Pathway

2000 ◽  
Vol 122 (4) ◽  
pp. 1161-1170 ◽  
Author(s):  
Jutta Papenbrock ◽  
Hans-Peter Mock ◽  
Ryouichi Tanaka ◽  
Elisabeth Kruse ◽  
Bernhard Grimm
Keyword(s):  
Biosynthetic Pathway ◽  
Magnesium Chelatase

scholarly journals The position of lysosomes within the cell determines their luminal pH

2016 ◽  
Vol 212 (6) ◽  
pp. 677-692 ◽  
Author(s):  
Danielle E. Johnson ◽  
Philip Ostrowski ◽  
Valentin Jaumouillé ◽  
Sergio Grinstein
Keyword(s):  
Fluorescence Microscopy ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Biosynthetic Pathway ◽  
Buffering Capacity ◽  
Ratiometric Fluorescence ◽  
Lysosomal Ph ◽  
Luminal Ph ◽  
Functional Versatility ◽  
Lysosomal Protein

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H+–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.

Localization of Adenosine Triphosphatase Activity in the Phleom of Nicotiana Tabacum

1972 ◽  
Vol 30 ◽  
pp. 230-231
Author(s):  
James Cronshaw ◽  
Jamison E. Gilder
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Higher Plants ◽  
High Surface ◽  
Root Tip ◽  
Animal Cells ◽  
Physiological Processes ◽  
Tip Cells ◽  
Atpase Localization

Adenosine triphosphatase (ATPase) activity has been shown to be associated with numerous physiological processes in both plants and animal cells. Biochemical studies have shown that in higher plants ATPase activity is high in cell wall preparations and is associated with the plasma membrane, nuclei, mitochondria, chloroplasts and lysosomes. However, there have been only a few ATPase localization studies of higher plants at the electron microscope level. Poux (1967) demonstrated ATPase activity associated with most cellular organelles in the protoderm cells of Cucumis roots. Hall (1971) has demonstrated ATPase activity in root tip cells of Zea mays. There was high surface activity largely associated with the plasma membrane and plasmodesmata. ATPase activity was also demonstrated in mitochondria, dictyosomes, endoplasmic reticulum and plastids.

A High Resolution Cytochemical Study of Nuclear ATPase in Human Spermatozoa

1975 ◽  
Vol 33 ◽  
pp. 594-595
Author(s):  
A. Sosa ◽  
L. Calzada
Keyword(s):  
High Resolution ◽  
Atpase Activity ◽  
Nuclear Membrane ◽  
Human Sperm ◽  
Sperm Nucleus ◽  
Human Spermatozoa ◽  
Cytochemical Study ◽  
Membrane Bound ◽  
Sperm Nuclei ◽  
Interchromatin Space

The dependence of nuclear metabolism on the function of the nuclear membrane is not well understood. Whether or not the function of the nuclear membrane is partial or totally responsible of the repressed template activity of human sperm nucleus has not at present been elucidated. One of the membrane-bound enzymatic activities which is concerned with the mechanisms whereby substances are thought to cross cell membranes is adenosintriphosphatase (ATPase). This prompted its characterization and distribution by high resolution photogrammetry on isolated human sperm nuclei. Isolated human spermatozoa nuclei were obtained as previously described. ATPase activity was demonstrated by the method of Wachstein and Meisel modified by Marchesi and Palade. ATPase activity was identified as dense and irregularly distributed granules confined to the internal leaflet of the nuclear membrane. Within the nucleus the appearance of the reaction product occurs as homogenous and dense precipitates in the interchromatin space.

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