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Comparison of the rat microsomal Mg-ATPase of various tissues
Abstract:
The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues.
Insights
Researchers identified a unique Mg-ATPase enzyme in rat tissues. This enzyme, found in plasma membranes, exhibits ATP-stimulated inactivation and hydrolyzes various nucleotide triphosphates, suggesting a significant role in cellular functions.
Area of Science:
- Biochemistry
- Cell Biology
- Enzymology
Background:
- Microsomal Mg-ATPase activity varies across rat tissues.
- Understanding the specific enzymes responsible for basal Mg-ATPase activity is crucial for cellular energy metabolism research.
Purpose of the Study:
- To characterize the properties of microsomal Mg-ATPase from different rat tissues.
- To identify the enzyme responsible for a significant portion of Ca-independent Mg-ATPase activity.
Main Methods:
- Sucrose gradient centrifugation was used to fractionate microsomal vesicles.
- Kinetic analysis, inactivation studies, and substrate specificity assays were performed.
- Inhibition studies with specific ATPase inhibitors and ionophores were conducted.
Main Results:
- The highest specific Mg-ATPase activity was localized in low-density vesicles containing plasma membrane.
- A significant fraction of microsomal Ca-independent Mg-ATPase showed ATP-stimulated inactivation, with a Km for ATP of 0.2 mM.
- The enzyme hydrolyzed various nucleotide triphosphates (CTP, GTP, UTP, ITP) but not non-nucleotide phosphocompounds, and required specific bivalent cations (Mg2+, Ca2+, Mn2+, Zn2+, Co2+).
Conclusions:
- A unique Mg-ATPase enzyme likely accounts for a major portion of basal microsomal Mg-ATPase activity in most rat tissues.
- This enzyme's properties, including ATP-stimulated inactivation and broad nucleotide specificity, differentiate it from other known ATPases.
- Further research is warranted to elucidate the physiological role of this unique Mg-ATPase.