Phosphatase is a kind of enzyme that cansubstrateDephosphorylationThe enzyme ofhydrolysisThe phosphate monoester removes the phosphate group on the substrate molecule and generatesPhosphate radicalIons and freehydroxyl。The role of phosphatase andkinaseThe kinase isphosphorylase, can use energy molecules, such asATP, add the phosphate group to the corresponding substrate molecule.A phosphatase commonly found in many organisms isalkaline phosphatase。
The induction and secretion of acid phosphatase is one of the important adaptive responses of plants to low phosphorus environment.Acid phosphatase can hydrolyze phosphate groups from different organic phosphorus substrates for plant absorption.Most plant acid phosphatases have no obvious substrate specificity. The substrates that can be hydrolyzed include RNA, DNA, 3-phosphoglycerate, hexose phosphate, etc.In vitro experiments, the activity of acid phosphatase purified from Arabidopsis and tomato was inhibited by the high concentration of Pi in the buffer solution. Further research found that Pi produced by acid phosphatase hydrolysis could negatively feedback the activity of most acid phosphatase.Acid phosphatase can also decompose some special organic phosphorus substrates, and the products can show special colors, such as 5-Bromo-4-chloro-3-indolyl phosphate (BCIP, blue), naphthalene ester phosphate（β-NaphthylAcid Phosphate，β-NAP/Fast black K, purple red), p-nitrophenol phosphate (pNPP, yellow).The activity of acid phosphatase can be judged by the color of reaction products.Using this method, mutants with abnormal responses to low phosphorus stress can be screened.The acid phosphatase induced by low phosphorus can be divided into two types: the acid phosphatase acting on the cell and the acid phosphatase secreted to the outside of the cell.The combination of the two ensures that plants can better cope with low phosphorus stress.The acid phosphatase in cells can recycle phosphorus in vivo through two ways: one is to convert organic phosphorus in plant vacuoles into Pi.Under normal circumstances, most of the phosphorus in plants is stored in vacuoles, and the content of Pi in cytoplasm is maintained within a certain range.When the plant is under low phosphorus stress, the Pi content in the plant body continuously decreases, the enzyme activity of acid phosphatase in the vacuole that is normally inhibited by high phosphorus environment recovers, the organic phosphorus stored in the vacuole is hydrolyzed and secreted to the cytoplasm through the phosphorus transport protein on the vacuole membrane, maintaining the dynamic balance of Pi content in the cytoplasm.Another way to realize phosphorus circulation in vivo is to reuse and transport phosphorus activation in aging tissues to young tissues.Robinson et al. reported that Arabidopsis purple acid phosphatase AtPAP26 was involved in the process of phosphorus reuse in aging tissues.The main function of secreted acid phosphatase is to decompose the organic phosphorus substrate in the soil environment and release Pi that can be directly absorbed and utilized by plants.Generally, secretory acid phosphatase is more stable than intracellular acid phosphatase.The pH activity range of secretory acid phosphatase (activity higher than 50%) is 4.0-7.6, and the temperature activity range (activity higher than 80%) is 22 ℃ - 48 ℃.This ensures that they can play a more efficient and sustainable role in complex soil media.It is reported that 80% of Pi that can be directly absorbed and utilized by plants in the soil comes from the decomposition of organic phosphorus substrate in the soil by acid phosphatase secreted into the extracellular space, which is enough to show the importance of acid phosphatase.
Secretory acid phosphatase can be divided into acid phosphatase released into environmental media and acid phosphatase attached to root surface according to its final role.Acid phosphatase secreted into the medium is relatively easier to study because it can be suspendedcell cultureOr seedling culture method, collectionLiquid mediumSecretory proteins in the.Different acid phosphatase were enriched, separated and identified by biochemical methods, and related genetic and physiological analysis was carried out.In recent years, through similar research methods, scientists have identified many acid phosphatase from a variety of different species, including white feather fan bean, kidney bean, tobacco, and Arabidopsis thaliana.However, the acid phosphatase attached to the root surface is relatively difficult to study, because it is difficult to obtain a large number of proteins for biochemical analysis, and only the genetic method is the most effective research method.Acid phosphatase attached to the root surface can be specifically detected by a synthetic organic phosphorus substrate BCIP.Cover the surface of Arabidopsis root with a solution containing 0.01% BCIP and 0.5% agar. After it is left overnight at room temperature, it can be seen that the surface of Arabidopsis root is dyed blue. The depth of blue reflects the activity of acid phosphatase on the surface of Arabidopsis root.BCIP staining of root surface is widely used in screening mutants related to low phosphorus response, such as pup1, pup2 and pup3 mutants were reported successively in 1998 and 2004, but unfortunately their respective mutant genes were not identified[1]。
Acid phosphatase activity and availability of organic phosphorus
Under phosphorus starvation, plants were induced to secrete acid phosphatase;On the other hand, the increase of acid phosphatase activity secreted by plant roots can hydrolyze and releaseOrganophosphorus compoundFor plant growth.In plants, acid phosphatase mainly accumulates in vacuoles. A large number of studies have shown that acid phosphatase plays a very important role in regulating phosphorus nutrition in plants, and it also plays a very important role in the metabolism and reuse of organic phosphorus. Its activity directly affects the availability of organic phosphorus.
In plants, the reuse of organic phosphorus by acid phosphatase is mainly achieved through two functions: 1) transforming organic phosphorus into inorganic phosphorus in plants;2) It transports phosphorus from senescent tissue to young tissue.Liang Hongling's research showed that under low phosphorus stress, the acid phosphatase activity of phosphorus efficient variety 97081 was higher than that of phosphorus inefficient variety 97009. Under low phosphorus conditions, the acid phosphatase activity of each part of 97081 increased significantly than that of the corresponding part of 97009. The grouping result of phosphorus in plants was also that the proportion of soluble phosphorus in the corresponding part of 97081 to total phosphorus was higher than that of 97009, which showed that 97081 under low phosphorus conditions,Acid phosphatase was strongly induced and its activity increased significantly.Phosphorus metabolism in 97081 is faster than that in 97009. Soluble phosphorus accounts for a high proportion of total phosphorus, which is more conducive to accelerating phosphorus transportation and promoting phosphorus reuse.Similar results have been reported in previous studies.It provides different forms of organic phosphorus sources for plants, and there are differences in plant growth, indicating that different organic phosphorus has different effectiveness on plants.Yadav et al. used three forms of organic phosphorus and soluble inorganic phosphorus, namely calcium magnesium phytate, lecithin and glycerol phosphate, as phosphorus sources to study nine cereal crops and oilseed crops, and found that the activity sequence of acid phosphatase secreted by crops at the same time was: phosphorus deficiency control>calcium magnesium phytate>phosphatidylcholine>glycerol phosphate>inorganic phosphorus.The activity of acid phosphatase increased with the difficulty of hydrolysis of organic phosphorus.When using organic phosphorus as phosphorus source to study the root exudation of APA by different crops, different results also appeared.For example, Adams et al. used phytate organic phosphorus and RNA as phosphorus sources to study the acid phosphatase activity secreted by the roots of two white lupin beans. They found that the treatments were arranged according to the acid phosphatase activity, Lupinus angust i folius L. The order was: RNA>phytate>no phosphorus application>inorganic phosphorus;Lupinus albus L. The order is: no phosphorus application>RNA>phytic acid>inorganic phosphorus, and the reason for this difference is still unclear.Therefore, under the condition of phosphorus deficiency, the acid phosphatase secreted by the root into the soil mainly hydrolyzes which form of organic phosphorus, and which form of organic phosphorus in the soil is more effective for plants need further research[2]。
Gene control of acid phosphatase secretion
Secreting acid phosphatase is a ubiquitous adaptive response of plants to low phosphorus stress, and this adaptive change is also the result of coordinated expression of phosphorus deficiency response genes. Through the direct or indirect effect of response gene products, it promotes the absorption, transport and effective utilization of phosphorus, and the research on acid phosphatase genes has made great progress.Goldstein et al. found that tomato roots can induce the production of secretory acid phosphatase under low phosphorus stress, and havegene expressionRegulation and series signal transmission system.Similar genes were also found in white feather fan bean. Low phosphorus stress can induce its enhanced expression, indicating that there may be a gene induced by low phosphorus stress that controls the secretion of acid phosphatase in plants.Some genes controlling acid phosphatase secretion have been located and isolated in plants.LAS AP1 is a 2187 bp long gene encoding acid phosphatase isolated from white feather fan bean. It contains a 1914 bp long coding frame and encodes a polypeptide composed of 637 amino acid residues. The amino acid sequence is the same as that of secreted acid phosphatase[2]。
alkaline phosphatase
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alkaline phosphatase(Alkaline phosphate, EC 3.1.3.1, AP) is a non-specific phosphate monoesterase, which can catalyze the hydrolysis of almost all phosphate monoesters, generate inorganic phosphoric acid and corresponding alcohols, phenols, sugars, etc., and also catalyze the transfer reaction of phosphate groups, and Escherichia coli A P is also a phosphite dependent hydrogenase.AP exists in almost all organisms except higher plants, can directly participate in phosphorus metabolism, and plays an important role in the digestion, absorption, secretion and ossification of calcium and phosphorus.In 1911, Levene et al. and Grosser et al. isolated (alkaline) phosphatase;In 1934, Davis proposed the name of alkaline phosphatase;In 1958, Ag ren et al. separated phosphoserine by isotope labeling;Schwartz also found this complex in Escherichia coli in 1961, and believed that serine might be a component of the active site of AP;In 1962, Plock et al. confirmed that AP was a metalloenzyme;In 1981, Bradshaw determined the complete amino acid sequence of Escherichia coli AP and cloned the gene phoA of Escherichia coli AP;Since then, the genes of various biological AP have been cloned one after another. In recent years, the research on the structure, mechanism and function of AP has become more in-depth, making the application of AP more extensive[3]。
application
AP is widely used in medicine, molecular biology and other fields.In clinical medicine, measuring the activity of AP in serum has become an important means to diagnose and monitor many diseases.AP is mainly used for obstructive jaundicePrimary liver cancer、Secondary liver cancer, cholestatic hepatitis, etc. When suffering from these diseases, hepatocytes overproduce AP and enter the blood through lymphatic channels and hepatic sinuses. At the same time, due to the obstruction of bile excretion in the intrahepatic biliary tract, the serum AP increases significantly.The obvious increase of blood midgut AP can be seen in various intestinal diseases, and there are also some literature reportsDigestive system diseases, autoimmune diseases andMalignant tumorIt can also appear in the patient's bloodimmunoglobulinComplex type AP, the mechanism of the emergence of this A P isoenzyme is not yet clear.As a sign of tumor tissue, AP isozyme is gradually recognized by people, such as PLA P in the serum of patients with malignant tumors such as lung, testis, ovary, pancreas, colon and lymph tissue.As a marker of abnormal bone metabolism, bone type AP has received more and more clinical attention;The quantitative determination of serum bone type A P activity can be used as an effective parameter to monitor the changes of bone formationOsteomalacia, rickets, etc.) and patients with early hyperthyroidism, chronic renal failure, and patients receiving kidney transplantation have different degrees of changes in the activity of bone type AP in their serum. The detection and dynamic observation of bone type AP activity will provide effective basis for early diagnosis of disease, monitoring of treatment effect, and prognosis of disease.
In terms of animal feeding and disease diagnosis, AP is an important biochemical indicator reflecting osteoblast activity, osteogenesis and calcium and phosphorus metabolism.The effects of insufficient supply of calcium and phosphorus on animals are mainly manifested as abnormal bone structure, osteomalacia, decreased appetite, growth retardation, and decreased production performance.The blood AP of young animals mainly comes from the bones. As the animals grow and mature and the bones mature, the AP from the bones gradually decreases.In animal nutrition research, serum AP activity is often used as an important biochemical indicator to help assess the suitability of dietary calcium and phosphorus levels.In animal disease diagnosis, according to the change rule of AP activity when osteoporosis and other bone diseases occur, serum AP activity can be used to diagnose bone diseases caused by calcium, phosphorus and VD imbalance.The detection of clinical bone type AP is more sensitive than the determination of calcium nutrition level in the body by blood calcium. Therefore, domestic and foreign studies agree that bone type AP is the most correct indicator reflecting the whole process of bone changes, and its specificity, sensitivity and accuracy are superior to the detection of other substances.In addition, when animals suffer from liver disease, gastrointestinal disease, kidney disease and zinc deficiency, serum AP will change. If we continue to study organ specificity, mechanism of A P change, and physiological function of AP in different animals, AP will have greater clinical significance in veterinary medicine.
In immunological research, AP labeled antibodies have been widely used for enzyme-linked immunofluorescence reaction (ELISA) and Western blot analysis, that is, the interaction of AP with chromogenic agents or depohosphorylated substrates that can emit light to reveal the existence of target and detection enzyme complexes. Compared with horseradish peroxidase, AP as a labeled enzyme has the advantages of high stability and sensitivity, while the disadvantages are high costMarking is difficult.In biochemistry and molecular biology, it is one of the conventional methods in gene cloning to remove the 5 '- terminal phosphate group of DNA molecule with AP catalyst to prevent the self ligation of vectors.Remove the 5 '- terminal phosphate group with AP, and then（γ-3 2P) A TP marks the 5 'end, which can be used for chemical sequencing, RNA sequencing and map construction of specific DNA or RNA fragments.AP was used in molecular hybridization instead of isotope labeled nucleotide probes.The most commonly used APs in research are: ① bacterial alkaline phosphatase (BAP); ②SAP (from an arctic shrimp); ③Calf intestinal alkaline phosphatase (CIAP); ④Placental alkaline phosphatase (PLAP) and secretory alkaline phosphatase (SEAP), the latter is the C-terminal deficient version of the former. Compared with PLAP, SEAP does not have the last 24 amino acids of the C-terminal of PLAP (these 24 amino acids constitute the region targeted to glycosylated phosphatidylinositol).In addition, the fusion of phoA gene with other genes to express hybrid proteins can be used for gene expression research.In 1995, it was also reported thatBiosensors。A common application in industry is based onPasteurizationAP can be destroyed, so AP is used as thePasteurizationLogo of[3]。
