AMPase

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Introductions

AMPase, also known as 5'-nucleotidase (5'-NT), is a hydrolase with low substrate specificity and can act on a wide range of nucleotides. This enzyme is mainly of hepatic origin and is widely found in human tissues such as liver, bile, intestine, brain, heart and pancreas. It is located on the cytoplasmic membrane, and in the liver this enzyme mainly exists in the bile duct and sinusoidal gap membrane. 5'-NT determination is mainly a colorimetric method, which is troublesome to operate and difficult to automate, but the reagents are easily available and suitable for the application in primary units.

Figure 1. Structure of AMPase.

Interference factors

• Determination with plasma can cause cloudy specimens, and anticoagulants chelated with metal ions can interfere with the activation of magnesium. Therefore, it is generally measured with serum.
• Anticoagulants that chelate with metal ions interfere with the activation of manganese ions.
• 5-'NT is a specific phosphate hydrolase that acts only on nucleoside-5'-phosphate such as AMP (adenosine-5'-phosphate or adenosine acid) to generate inorganic phosphate and nucleoside, which is hydrolyzed by all four 5-ribonucleoside-phosphate. This enzyme is widely present in the cell membranes of various human tissues, and its optimum pH is 6.6-7.0. It is activated by Mg²⁺ but inhibited by Ni²⁺.

Source

5'-nucleotidase is widely distributed in liver, bile, pancreas, intestine, heart, brain, lung, kidney, pituitary, thyroid, prostate, testis and other organs and tissues, localized on cell membranes, and the hepatobiliary system is the main source of 5'-nucleotidase in serum. In the liver, it is mainly found in the bile ducts and sinusoidal spaces.

Detection method

• Molybdenum blue colorimetric method
• Chemiluminescence method
• Enzyme method
• Coupling rate method

Principle of detection

5'-Nucleotidase (5'-NT) catalyzes the hydrolysis of 5'-hypoxanthine nucleotide to produce hypoxanthine and ammonia, hypoxanthine is generated by purine nucleoside phosphorylase (PNP), hypoxanthine is generated by xanthine oxidase (XOD) to produce uric acid and hydrogen peroxide (H₂0₂), H₂0₂and 4-aminoantipyrine (4-AA) and T00S by peroxidase (POD). H₂0₂ was combined with 4-Aminoantipyrine (4-AA) and T00S to produce awake imino compounds by the action of peroxidase (POD), and the 5' -NT activity in the samples was determined by measuring the change in absorbance at the corresponding wavelengths.

Membrane-bound and soluble forms

Studies of mammalian 5'-nucleotidases have shown that four different forms of 5'-nucleotidase exist: a membrane-bound form and three soluble forms. The membrane-bound form is anchored to the plasma membrane by a GPI at the C-terminus. One of the soluble forms appears to be derived from the GPI-anchored ex-5'-nucleotidase and has an extracellular location. The two cytoplasmic forms of the enzyme have similar characteristics, but can be distinguished on the basis of their preferential affinity for nucleotide substrates. the GPI-anchored form exists as a dimer, with the two subunits linked by disulfide bonds. The soluble form can exist as a dimer or a tetramer. Usually at least 50% of the enzyme is present in surface bound form

Clinical significance

Serum 5'-NT assay is mainly used for the diagnosis of hepatobiliary system diseases and differential diagnosis of skeletal diseases. Increased serum 5'-NT activity is mainly seen in diseases of the hepatobiliary system, such as obstructive jaundice, primary and secondary hepatocellular carcinoma, and hepatitis, and its activity usually changes in line with the activity of ALP. In diseases of the skeletal system, such as tumor metastasis, deformational osteitis, hyperparathyroidism, and rickets, ALP activity is usually increased, but 5'-NT is normal. Therefore, simultaneous measurement of ALP and 5'- NT is useful for the differential diagnosis of diseases of the hepatobiliary and skeletal systems.