CN105695558A - Kit for detecting enzyme activity of PEMT (phosphatidyl ethanolamine N-methyltransferase) - Google Patents
Kit for detecting enzyme activity of PEMT (phosphatidyl ethanolamine N-methyltransferase) Download PDFInfo
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Abstract
The invention provides a combined product for detecting enzyme activity of PEMT (phosphatidyl ethanolamine N-methyltransferase). The combined product contains S-adenosine-L-methionine or an enzyme reaction system capable of producing S-adenosine-L-methionine, a methyl receptor and S-adenosine-L-homocysteine hydrolase, wherein the enzyme reaction system capable of producing S-adenosine-L-methionine comprises adenosine triphosphate, methionine and S-adenosylmethionine. Preferably, the combined product adopts a kit form, and all components are in self-existent reagent states. The combined product can be used on semi-automatic or full-automatic analysis detection equipment and is high in detection sensitivity, high in specificity and convenient to operate, so that the combined product can be popularized and used actually.
Description
Technical field
The invention belongs to biomedicine technical field, belong to again clinical diagnosis and detection field simultaneously。Specifically the invention provides the combination product of a kind of detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase (PEMT, PhosphatidylethanolamineN-methyltransferase, EC2.1.1.17) enzyme activity。
Background technology
Choline is a kind of strong organic base, is the constituent of lecithin, exists among sphingomyelin, is a kind of nutrient substance required for life entity, and liver and the 26S Proteasome Structure and Function of brain, lipid metabolism and fetal development are had important function by choline。Fat is had affinity by choline; fat can be made to be transferred out by blood by liver with phospholipid form; thus preventing the formation of fatty liver; the hepatic fibrosis that effectively protection and treatment ethanol cause; there is the effect of anti-liver lipid peroxide, thus reducing the hepatocellular apoptosis that ethanol causes, regulating hepatocyte activity; the cytotoxicity and the enhancing interleukin-22 2 that reduce tumor necrosis factor protect hepatocellular effect, and choline also has the effect that protection muscular tissue avoids damage to simultaneously。Choline is most important to the growth of fetus, and it affects the increment of stem cell and apoptosis and changes the 26S Proteasome Structure and Function of brain, and same choline shortage can increase the risk suffering from neural tube defect。
In mankind's body, choline can partly come from phosphatidylcholine, and phosphatidylcholine is the ultimate constituent of all eukaryotic cell membrane。PHOSPHATIDYL ETHANOLAMINE N-transmethylase (PEMT) is a kind of catalyzing enzyme of PHOSPHATIDYL ETHANOLAMINE N-methylation reaction in catalysis phospholipid metabolism approach, in mammal, the methyl group of S-adenylyl-METHIONINE can be transferred to PHOSPHATIDYL ETHANOLAMINE (phosphatidylethanolamine by this enzyme, PE) N end, by intermediate state phospholipids acyl N-monomethyl-ethanolamine (PME) of two PHOSPHATIDYL ETHANOLAMINE and phosphatidyl N, N-dimethylethanolamine (PDE), final synthetic phospholipid phatidylcholine (phosphatidylcholine, PC)。
PMET is extremely low in the activity of extrahepatic tissue, mainly expresses in normal hepatic tissue, and in mammal body, liver is the organ uniquely with notable PEMT enzyme activity。PEMT has two types, is PEMT1 and PEMT2 respectively, and wherein PEMT1 is positioned at the surface of the kytoplasm side of endoplasmic reticulum, has very high total PEMT vigor, is the enzyme of main synthetic phospholipid phatidylcholine in liver。PEMT2 is distributed in the film part that mitochondrion is relevant, find according to the study, PEMT2 is responsible for the synthesis of hepatocyte 20%-40% phosphatidylcholine, existing research proves that its gene expression and hepatocellular increment division are in negative correlation, in fast the cell such as hepatoma carcinoma cell, regenerating hepatic tissue of rising in value, the expression of PEMT2 is very low, and in normal liver cell, the expression of PEMT and activity are all higher, and research shows, the mice of PEMT gene knockout suffers from fatty liver, and is more susceptible to suffer from the hepatocarcinoma of chemical induction。Also studies have found that the process LAN of PEMT2 can suppress the increment of Transfected Recombinant Plasmid rat liver cancer CBRH-7919 cell the apoptosis of inducing cell。
PEMT gene has significantly high polymorphism, has identified more than 100 SNP at present, and wherein 5465G → A and 744G → C is the known SNP with function, it is possible to affect the demand of the activating agent choline of albumen and the health of human body。Non-alcoholic fatty liver disease (nonalcoholicfattyliverdisease, NAFLD) is one of common chronic disease, and the sickness rate of crowd is up to 25%, and NAFLD may develop into hepatic necrosis, fibrosis even liver cirrhosis。In PEMT gene, the result of 5465G → A is to cause that 175 amino acids are by replacing, thus causing code segment PEMT activity to lack, this gene pleiomorphism is equivalent to 1.7 times of normal person in NAFLD patient's incidence rate, may infer that the morbidity of PEMT activity disappearance and non-alcoholic fatty liver disease has direct relation, therefore speculate that mankind's non-alcoholic fatty liver disease is relevant with PEMT gene pleiomorphism。744G → C is positioned at the promoter region of this gene, there are about 50 bases and be positioned at estrogen response element region, 744G → C can affect the expression of this gene, by the sensing of the PEMT gene of estrogen-mediated thus increasing the sensitivity that choline is lacked by women, there is research display 744CC and GG to compare and adding the risk suffered from breast cancer。
There is the PEMT vigor of research display Parkinsonian's brain volume cortex lower than normal person, the methylation level of brain can be dramatically increased after exogenous increase PME and PDE, but do not obtain any correction in volume cortex, illustrate that parkinson disease are relevant with the function of PEMT, have bibliographical information PEMTV175M relevant to the scattered onset Alzheimer disease (Alzheimer'sDisease) of Chinese population。
Also have and study display, in Mice Body, lack PEMT expression can cause high density lipoprotein (highdensitylipoproteins, HDL) reduction measured and very low density lipoprotein (VLDL) (verylowdensitylipoproteins, and low density lipoprotein, LDL (lowdensitylipoproteins VLDL), LDL) rising measured, the rising of VLDL and LDL content is the risk factor that atherosclerosis occurs。PEMT expresses one of reduction inducement being likely to be human atherosclerosis with activity as can be seen here。
The detection method of current PEMT enzyme activity is radiochemical method, namely by detecting the amount generating [methyl-3H] that phospholipid (phospholipids) combines with S-adenosine-L-(methyl-3H)-methionine for substrate, measure the activity of PEMT, the early stage of this radioactive method processes more complicated, technology required higher and easily causes pollution, being unfavorable for promoting the use of。
Summary of the invention
Therefore, defect based on above-mentioned prior art, in order to solve existing PHOSPHATIDYL ETHANOLAMINE N-transmethylase (PEMT, PhosphatidylethanolamineN-methyltransferase, EC2.1.1.17) problem that enzyme activity detection method is not easily promoted, it is an object of the invention to provide a kind of quick, special, detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase (PEMT accurately and reliably, PhosphatidylethanolamineN-methyltransferase, EC2.1.1.17) combination product of enzyme activity。Adopting this combination product can use on semi or fully automated analysis detection equipment, and detection sensitivity is high, specificity is high, easy and simple to handle, thus can obtain practical promoting the use of。
For foregoing invention purpose, the present invention is achieved through the following technical solutions:
Specifically, the present invention provides a kind of combination product detecting PEMT enzyme activity, wherein, described combination product contains: the enzymatic reaction system of SAMe (SAM) or generation SAMe, the enzymatic reaction system of described generation SAMe includes adenosine triphosphate, methionine and S-adenosylmethionine synzyme；Acceptors；S-adenosine-L-homocysteine hydrolase (SAHase)。Preferably, described combination product is kit form, and wherein respectively comprising composition is the reagent state being individually present。
Preferably, the combination product according to aforesaid detection PEMT enzyme activity, wherein, described acceptors is cytoskeletal protein compound。Preferably, described cytoskeletal protein compound is PHOSPHATIDYL ETHANOLAMINE (PE) or phosphatidyl N-monomethyl-ethanolamine (PME)。It most preferably is PHOSPHATIDYL ETHANOLAMINE。
It is highly preferred that the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product also includes buffer。The pH value of system can be cushioned by generally accepted in physics, chemistry and zymetology or stablize the reagent in the scope of 6.5～10.0 by described buffer, the pH value of the catalytic reaction of PHOSPHATIDYL ETHANOLAMINE N-transmethylase can be effectively stablized in its existence, and the physico-chemical property of PHOSPHATIDYL ETHANOLAMINE N-transmethylase can be stablized, thus being beneficial to analysis and the detection of the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase。It includes following instance but is not limited to these examples:
Glycine-NaOH: pH8.6～10.6
Citric acid/sodium citrate: pH3.0～6.6
Hydrophosphate/citric acid (salt): pH2.2～8.0
Phosphate: pH4.9～8.2
2-morpholino ethanesulfonic acid buffer (MES): pH5.5～6.7
3-morpholine propane sulfonic acid buffer (MOPS): pH6.5～7.9
4-hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES): pH6.8～8.2
Tris-hydrochloric acid: pH7.1～8.9
Boric acid-borate buffer solution: pH7.4～9.0
Barbital sodium-hydrochloride buffer: pH6.8～9.6
Most preferably, described buffer is Tris-hydrochloric acid。
More preferably, the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product includes: described combination product pH ranges for 6.5～10.0, it is preferred to 7.5～8.9, it is most preferred that be 8.5；It is 10～1000mmol/L that described combination product contains described buffer, it is preferred to 25～100mmol/L, it is most preferred that for 30mmol/L；Described acceptors is 0.002～10mmol/L, it is preferred to 0.05～0.5mmol/L, it is most preferred that for 0.2mmol/L；Described SAMe is 0.02～2mmol/L, it is preferred to 0.05～0.5mmol/L, it is most preferred that for 0.1mmol/L；And described S-adenosine-L-homocysteine hydrolase is 0.01～100KU/L, it is preferred to 0.1～50KU/L, it is most preferred that for 10KU/L。
Preferably, according to the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product is possibly together with reagent, and described reagent is oleic acid, cholate, Tween20, Brij35 or sodium taurocholate。Preferably, the concentration expressed in percentage by volume of described reagent is 0.05-1%。It most preferably is 0.5%。
It is highly preferred that according to the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product is dry powder or liquid。
More preferably, according to the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product is double; two agent reagent or three doses of reagent。
Specifically, the combination product of aforesaid detection PEMT enzyme activity, wherein, described combination product includes:
Experiments show that, consider from economy two aspect of the accuracy of measurement result and preparation cost, no matter be double; two agent or three doses, following composition relation is ideal。
It is therefore preferred that the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product includes:
Most preferably, combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product pH is 8.5, described combination product contains described buffer 30mmol/L, described acceptors 0.2mmol/L, described SAM0.1mmol/L, described S-adenosine-L-homocysteine hydrolase 10KU/L。
Preferably, the combination product according to aforesaid detection PEMT enzyme activity, wherein, described combination product can be made into following double; two agent reagent:
Reagent 1
Buffer, SAM
Reagent 2
Buffer, acceptors, S-adenosine-L-homocysteine hydrolase
Reagent can be formed into dry powder, uses after dissolving；Or it is made into liquid reagent, it is possible to directly use。
Above-mentioned pair of agent reagent can also be made into following three doses of reagent:
Reagent 1
Buffer, SAM
Reagent 2
Buffer, acceptors
Reagent 3
Buffer, S-adenosine-L-homocysteine hydrolase
Reagent can be formed into dry powder, uses after dissolving；Or it is made into liquid reagent, it is possible to directly use。
Present invention also offers the application in the product prepared for analyzing the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase in sample or apparatus of the combination product of aforementioned detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity。Preferably, described sample is selected from one or more in biological tissue, body fluid and cell。It is highly preferred that described biological tissue is selected from one or more in lung, colon, ovary, Placenta Hominis, pancreas, thymus, small intestinal, kidney, brain and liver, described body fluid is blood。
Present invention also offers the combination product of aforementioned detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity in preparation for the application in the product of the abnormal disease caused of the enzyme activity analyzing, detect or diagnosing the PHOSPHATIDYL ETHANOLAMINE N-transmethylase in sample to be tested, described disease is preferably by the abnormal heredity caused of the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase and metabolic disease。Or in preparation for the application in the product of Clinical detection or the enzyme activity relevant disease of diagnosis and PHOSPHATIDYL ETHANOLAMINE N-transmethylase。
Present invention also offers the combination product of aforementioned detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity and prepare the application in the product for instructing the enzyme activity related drugs with PHOSPHATIDYL ETHANOLAMINE N-transmethylase to use。
The detection method of detection PEMT enzyme activity combination product of the present invention includes:
(1) contact to form the methylate of S-adenosine-L-homocysteine (SAH) and acceptors with SAMe (SAM) and acceptors by the sample containing PEMT,
(2) by described SAH and S-adenosine-L-homocysteine hydrolase (SAHase) contacts to generate homocysteine (Hcy) and adenosine (Ado), photometer is adopted to assess the generation speed of described Ado to detect the enzyme activity of PEMT described in described sample。
Preferably, according to aforesaid PEMT enzyme activity detection method, wherein, described acceptors is cytoskeletal protein compound。Preferably, described cytoskeletal protein compound is PHOSPHATIDYL ETHANOLAMINE (PE) or phosphatidyl N-monomethyl-ethanolamine (PME)。It most preferably is PHOSPHATIDYL ETHANOLAMINE。
It is highly preferred that according to aforesaid PEMT enzyme activity detection method, wherein, described method includes:
(1) described sample containing PEMT, described SAM and described acceptors mixed and react acquisition reactant liquor 1；
(2) add in described SAHase in the described reactant liquor 1 in step (1) and react, it is thus achieved that reactant liquor 2；
(3) supernatant is obtained by centrifugal for the reactant liquor 2 described in step (2)；
(4) adopt the absorbance value of the supernatant described in photometer detecting step (3), cither indirectly or directly assess the generation speed of described Ado to detect the enzyme activity of phosphatidyl PEMT described in described sample。
More preferably, according to aforesaid PEMT enzyme activity detection method, wherein, described method includes: add buffer in step (1) process and step (2) process。Preferably, described buffer is selected from one or more in glycine/sodium hydroxide, citric acid/sodium citrate, hydrophosphate/citric acid (salt), phosphate, 2-morpholino ethanesulfonic acid buffer, 3-morpholine propane sulfonic acid buffer, 4-hydroxyethyl piperazine ethanesulfonic acid buffer, Tris-hydrochloric acid, boric acid-Borax, barbital sodium-hydrochloride buffer。It most preferably is Tris-hydrochloric acid。
It is further preferred that according to aforesaid PEMT enzyme activity detection method, wherein, described buffer is 10～1000mmol/L, it is preferred to 25～100mmol/L, it is most preferred that for 30mmol/L。Described acceptors is 0.002～10mmol/L, it is preferred to 0.05～0.5mmol/L, it is most preferred that for 0.2mmol/L。Described SAMe is 0.02～2mmol/L, it is preferred to 0.05～0.5mmol/L, it is most preferred that for 0.1mmol/L。And described S-adenosine-L-homocysteine hydrolase is 0.01～100KU/L, it is preferred to 0.1～50KU/L, it is most preferred that for 10KU/L。
Or it is further preferred that according to aforesaid PEMT enzyme activity detection method, wherein, the condition of reaction described in step (1) bathes 60min for being placed in 35 DEG C of environment temperature。Preferably, the condition of reaction described in step (2) bathes 10min for being placed in 30 DEG C of environment temperature。It is highly preferred that being centrifuged described in step (3) is 10,000g centrifugal 10 minutes。
Or preferably; according to aforesaid PEMT enzyme activity detection method; wherein; described method is additionally included in that step (1) is front carries out early stage process by the described sample containing PEMT; described early stage processes and mixes with reagent for the described sample containing PEMT, and described reagent is oleic acid, cholate, Tween20, Brij35 or sodium taurocholate。Preferably, described reagent is after above-mentioned mixing, and the concentration expressed in percentage by volume reached is 0.05-1%。It most preferably is 0.5%。
It is further preferred that according to aforesaid PEMT enzyme activity detection method, wherein, described method also includes, after described early stage processes, the described sample containing PEMT is placed on ice 30min。
Preferably, according to aforesaid PEMT enzyme activity detection method, wherein, the enzymatic reaction system that described SAM is made up of adenosine triphosphate, methionine and S-adenosylmethionine synzyme (EC2.5.1.6) is generated。
Preferably, according to aforesaid PEMT enzyme activity detection method, wherein, described sample is selected from one or more in biological tissue, body fluid and cell。Preferably, described biological tissue is selected from one or more in lung, colon, ovary, Placenta Hominis, pancreas, thymus, small intestinal, kidney, brain and liver, and described body fluid is blood。
Specifically, the present invention provides a kind of PEMT enzyme activity detection method, and its basic implementation process is as follows:
PEMT can utilize SAMe (SAM) as methyl donor, the methyl of SAM is transferred on acceptors, generate the methylate of acceptors, SAM is converted into S-adenosine-L-homocysteine (SAH) simultaneously, SAH is at S-adenosine-L-homocysteine hydrolase (SAHase, EC3.3.1.1) adenosine (Ado) is produced under effect, the speed of Ado produced is directly proportional to the minimizing speed of SAM, and the generation speed assessing described Ado can detect the enzyme activity of PEMT。Described acceptors is cytoskeletal protein compound, such as PHOSPHATIDYL ETHANOLAMINE (PE), phosphatidyl N-methylethanolamine (PME), it is preferred to PHOSPHATIDYL ETHANOLAMINE。
Can directly assess the generation speed of described Ado。Absorb as Ado has specificity light when ultraviolet specific wavelength, assess described Ado generating rate can the minimizing speed of indirect assessment SAM, and then calculate the enzyme activity of PEMT in sample to be tested。
PEMT enzyme activity detection method of the present invention, carries out early stage and processes the PEMT enzymatic activity that can be obviously enhanced sample sample。Further, PEMT enzyme activity detection method of the present invention, the reagent that sample carries out early stage process can be oleic acid, cholate, Tween20, Brij35, sodium taurocholate etc.。Further, PEMT enzyme activity detection method of the present invention, sample is carried out the suitableeest scope 0.05-1% of concentration of early stage reagent treatment。
The principle of PEMT enzyme activity detection method of the present invention can simplify such as Fig. 1。
The combination product of detection PEMT enzyme activity of the present invention may be used for the abnormal various diseases caused of enzyme activity of the PHOSPHATIDYL ETHANOLAMINE N-transmethylase analyzed, detect and diagnose in sample to be tested, particularly by the abnormal various heredity caused of the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase and metabolic disease。Such that it is able to be widely applied to Clinical detection or diagnose the disease relevant to the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase；And this PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity detection method can be additionally used in reagent and instructs the drug use relevant to the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase。
In the combination product of detection PEMT enzyme activity of the present invention, described substrate SAMe can be synthetic compound；Further, it is also possible to the enzymatic reaction system being made up of adenosine triphosphate, methionine and S-adenosylmethionine synzyme (EC2.5.1.6) is generated。
In the combination product of detection PEMT enzyme activity of the present invention, described S-adenosine-L-homocysteine hydrolase (EC3.3.1.1) can be various source of species, it includes but not limited to it is natural extract, or it is recombinant expressed, or the enzyme through transformation or modification, as long as it has catalysis S-adenosine-L-homocysteine (SAH) hydrolysis produces the function of adenosine, it it is all the category contained of the present invention。
In the combination product of detection PEMT enzyme activity of the present invention, described S-adenosylmethionine synzyme (EC2.5.1.6) can be various source of species, it includes but not limited to it is natural extract, or it is recombinant expressed, or the enzyme through transformation or modification, as long as it has a catalysis adenosine triphosphate and methionine reaction generates the function of SAMe (SAM), it it is all the category contained of the present invention。
The detection PEMT enzyme activity combination product of the present invention has but is not limited to following beneficial effect:
(1) compared with prior art radiochemical method, the combination product early stage of the detection PEMT enzyme activity of the present invention processes simple, technology is less demanding and not easily pollute。
(2) known based on the enzyme activity determination in embodiment, the combination product of the detection PEMT enzyme activity of the present invention can use on semi or fully automated analysis detection equipment, and detection sensitivity is high, specificity is high, not by the pollution of interior allogenic material and interference, it is simple to be applied to analysis and the detection of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity。
Accompanying drawing explanation
Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:
Fig. 1 illustrates the detection method principle of detection PEMT enzyme activity combination product of the present invention。
Detailed description of the invention
The present invention is further illustrated below by specific embodiment, it should be understood, however, that, these embodiments are only used for the use specifically described in more detail, and are not to be construed as limiting in any form the present invention。
In following example, except the test material particularly pointed out, condition and operational approach, the many materials used in example and operational approach are well known in the art。Therefore, it will be apparent to those skilled in the art that within a context, if not specified, material therefor of the present invention and operational approach are well known in the art。
Following PE, SAM, S-adenosine-L-homocysteine hydrolase is all purchased from SIGMA-ALDRICH, and using detection equipment is 752 type ultraviolet-uisible spectrophotometers。
Embodiment 1: the thick extraction of rat liver endoplasmic reticulum
Weigh the rat liver 20g wiping out fatty tissue, add the sucrose solution of the 0.25M (including 0.01M, pH8 phosphate buffer) of 200mL, with glass homogenizer homogenate 20 times, Sigma3-30K centrifuge is centrifuged 10 minutes with 2600-2800rpm, after four layers of filtered through gauze, abandons precipitation。Supernatant is centrifuged 30 minutes with 15000rpm, and gained precipitation is the part containing endoplasmic reticulum, is suspended in the sucrose solution of 0.25M (including 0.01M, pH8.0 phosphate buffer), and endoplasmic reticulum can preserve under-80 DEG C of conditions。
Embodiment 2: Hepar Mus endoplasmic reticulum PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity determination (carries out
Early stage processes)
The PEMT detectable of the present embodiment is three doses of reagent, including
Reagent 1
Tris-hydrochloride buffer (pH8.5) 30mmol/L
SAM0.1mmol/L
Reagent 2
Tris-hydrochloride buffer (pH8.5) 30mmol/L
PE0.2mmol/L
Reagent 3
Kaliumphosphate buffer (pH8.0) 30mmol/L
S-adenosine-L-homocysteine hydrolase 10KU/L
The sample of tested PEMT is the rat liver endoplasmic reticulum extracted, and after break process, adds sodium cholate solution, make final volume percentage concentration reach 0.5% in sample, gently after mixing, places 30min on ice stand-by。In reaction system, total protein consumption is 10ug, and reaction system is 250ul。Making 5 parts of parallel sampless altogether, this detection method adopts end-point method detection PEMT enzyme activity。
Detection method:
Comparison: reagent 1 is mixed with the sample through inactivation treatment with 2 that to be placed in 35 DEG C of environment warm bathes 60 minutes, it is subsequently adding reagent 3 and is placed in 30 DEG C of environment temperature bath 10 minutes, terminate reaction afterwards, mixed liquor after termination is through 10,000g takes supernatant after centrifugal 10 minutes and joins in spectrophotometer, detect the absorbance value of product when 260nm, be designated as A0。
Sample: reagent 1 is mixed with sample with 2 and is placed in 35 DEG C of environment temperature bath 60 minutes, it is subsequently adding reagent 3 and is placed in 30 DEG C of environment temperature bath 10 minutes, terminate reaction afterwards, mixed liquor after termination is through 10,000g takes supernatant after centrifugal 10 minutes and joins in spectrophotometer, detect the absorbance value of product when 260nm, be designated as A1。
The vigor light absorption value Δ A=A of PEMT1-A0。
Enzyme activity unit is: the nanomole number of every milligram of albumen conversion of substrate per hour, represents with nmol/h/mg albumen。
Experimental result A0Value is 0.476 ± 0.006, A1Value is 1.109 ± 0.032, Δ A average is 0.632, changing into enzyme activity average is 369.70nmol/h/mg albumen, it can be seen that the method for the detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity of the present invention and reagent can effectively detect the PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity in sample and measure stable。
Embodiment 3: Hepar Mus endoplasmic reticulum PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity determination is (to sample
Originally carry out early stage process to compare)
The PHOSPHATIDYL ETHANOLAMINE N-transmethylase detectable of the present embodiment is three doses of reagent, including
Reagent 1
Tris-hydrochloride buffer (pH8.5) 30mmol/L
SAM0.1mmol/L
Reagent 2
Tris-hydrochloride buffer (pH8.5) 30mmol/L
PE0.2mmol/L
Reagent 3
Kaliumphosphate buffer (pH8.0) 30mmol/L
S-adenosine-L-homocysteine hydrolase 10KU/L
The sample of tested PHOSPHATIDYL ETHANOLAMINE N-transmethylase is the rat liver endoplasmic reticulum extracted, and after break process, is divided into two parts, and a copy of it adds sodium cholate solution in sample, makes final concentration reach 0.5%, gently after mixing, places 30min on ice；Another part adds the sample suspensions liquid of equivalent, places 30min on ice gently after mixing, and in reaction system, total protein consumption is 10ug, and reaction system is 250ul。This detection method adopts end-point method detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity。
Detection method: identical with detection method in embodiment 2。
Comparison of experiment results:
PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity after early stage process is 341.65nmol/h/mg albumen, the PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity that non-premenstruum processes is 215.07nmol/h/mg albumen, use the reagent in the present invention that sample carries out early stage process as can be seen here and can significantly improve PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity in sample, and then improve the sensitivity of the method for described detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity。The method of the detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity of the present invention and reagent can effectively detect the PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity in sample and measure stable。
Although at this, the present invention is carried out a degree of description, it will be apparent that, without departing from spirit and scope of the invention when, one of ordinary skill in the art can carry out the suitable change of each condition。Summarizing and instantiation it is understood that the invention is not restricted to described embodiment, its right is attributed to scope of the claims, and includes the equivalent replacement of described each factor。
Claims (10)
1. the combination product detecting PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity, it is characterised in that described combination product contains:
The enzymatic reaction system of SAMe or generation SAMe, the enzymatic reaction system of described generation SAMe includes adenosine triphosphate, methionine and S-adenosylmethionine synzyme；
Acceptors；
S-adenosine-L-homocysteine hydrolase；
Preferably, described combination product is kit form, and wherein respectively comprising composition is respective self-existent reagent state。
2. detect the combination product of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity according to claim 1, it is characterised in that described acceptors is cytoskeletal protein compound；Preferably, described cytoskeletal protein compound is PHOSPHATIDYL ETHANOLAMINE or phosphatidyl N-monomethyl-ethanolamine；It most preferably is PHOSPHATIDYL ETHANOLAMINE。
3. detect the combination product of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity according to claim 2, it is characterised in that described combination product also includes buffer；
Preferably, described buffer is selected from one or more in glycine/sodium hydroxide, citric acid/sodium citrate, hydrophosphate/citric acid (salt), phosphate, 2-morpholino ethanesulfonic acid buffer, 3-morpholine propane sulfonic acid buffer, 4-hydroxyethyl piperazine ethanesulfonic acid buffer, Tris-hydrochloric acid, boric acid-Borax and barbital sodium-hydrochloride buffer；
Most preferably, described buffer is Tris-hydrochloric acid。
4. detect the combination product of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity according to claim 3, it is characterised in that:
Described combination product pH ranges for 6.5～10.0, it is preferred to 7.5～8.9, it is most preferred that be 8.5；
Described buffer is 10～1000mmol/L, it is preferred to 25～100mmol/L, it is most preferred that for 30mmol/L；
Described acceptors is 0.002～10mmol/L, it is preferred to 0.05～0.5mmol/L, it is most preferred that for 0.2mmol/L；
Described SAMe is 0.02～2mmol/L, it is preferred to 0.05～0.5mmol/L, it is most preferred that for 0.1mmol/L；And
Described S-adenosine-L-homocysteine hydrolase is 0.01～100KU/L, it is preferred to 0.1～50KU/L, it is most preferred that for 10KU/L。
5. according to any one of Claims 1-4, detect the combination product of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity; it is characterized in that; described combination product is possibly together with reagent, and described reagent is oleic acid, cholate, Tween20, Brij35 or sodium taurocholate；Preferably, the concentration expressed in percentage by volume of described reagent is 0.05-1%；It most preferably is 0.5%。
6. according to any one of claim 1 to 5, detect the combination product of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity, it is characterised in that described combination product is dry powder or liquid。
7. according to any one of claim 1 to 6, detect the combination product of PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity, it is characterised in that described combination product is double; two agent reagent or three doses of reagent。
8. the combination product of detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity according to any one of claim 1 to 7 is used for analyzing the application in the product of the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase in sample or apparatus in preparation；
Preferably, described sample is selected from one or more in biological tissue, body fluid and cell；
It is highly preferred that described biological tissue is selected from one or more in lung, colon, ovary, Placenta Hominis, pancreas, thymus, small intestinal, kidney, brain and liver, described body fluid is blood。
9. the combination product of detection PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity according to any one of claim 1 to 7 is in preparation for the application in the product of the abnormal disease caused of the enzyme activity analyzing, detect or diagnosing the PHOSPHATIDYL ETHANOLAMINE N-transmethylase in sample to be tested, and described disease is preferably the heredity and metabolic disease that are extremely caused by the enzyme activity of PHOSPHATIDYL ETHANOLAMINE N-transmethylase；Or
In preparation for the application in the product of Clinical detection or the enzyme activity relevant disease of diagnosis and PHOSPHATIDYL ETHANOLAMINE N-transmethylase。
10. the combination product detecting PHOSPHATIDYL ETHANOLAMINE N-transmethylase enzyme activity according to any one of claim 1 to 7 is used for the application in the product instructing the enzyme activity related drugs with PHOSPHATIDYL ETHANOLAMINE N-transmethylase to use in preparation。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249342A (en) * | 1998-09-28 | 2000-04-05 | 上海新黄浦复旦基因工程有限公司 | Human phosphatidyl ethanolamine-N-methyltransferase, its coding sequence and its preparing process |
| CA2477909A1 (en) * | 2002-03-11 | 2003-09-25 | Lipomics Technologies, Inc. | Novel metabolic targets and markers |
| CN102066569A (en) * | 2008-06-20 | 2011-05-18 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and a method for making the same |
| WO2011112245A1 (en) * | 2010-03-10 | 2011-09-15 | Albert Einstein College Of Medicine Of Yeshiva University | Luciferase-linked analysis of dna-methyltransferase, protein methyltransferase and s-adenosylhomocysteiene and uses thereof |
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2016
- 2016-03-10 CN CN201610137000.5A patent/CN105695558A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249342A (en) * | 1998-09-28 | 2000-04-05 | 上海新黄浦复旦基因工程有限公司 | Human phosphatidyl ethanolamine-N-methyltransferase, its coding sequence and its preparing process |
| CA2477909A1 (en) * | 2002-03-11 | 2003-09-25 | Lipomics Technologies, Inc. | Novel metabolic targets and markers |
| CN102066569A (en) * | 2008-06-20 | 2011-05-18 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and a method for making the same |
| WO2011112245A1 (en) * | 2010-03-10 | 2011-09-15 | Albert Einstein College Of Medicine Of Yeshiva University | Luciferase-linked analysis of dna-methyltransferase, protein methyltransferase and s-adenosylhomocysteiene and uses thereof |
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