JPS615800A - Determination of protein binding with vitamin d - Google Patents

Abstract

PURPOSE:In the determination of the title substance existing in body fluid of mammarians, a sample to be measured is combined with monomer-type actin, deoxyribonuclease I and deoxyribonucleic acid to effect determination whereby simple and high-accuracy measurement becomes possible. CONSTITUTION:A sample to be determined, which originates from mammarians and contains proteins binding with vitamin D is combined with an excessive amount of monomer-type actin to form a complex of vitamin D-binding protein- monomer-type actin. The rest of the actin is converted into polymer-type actin. The resultant reaction system is mixed with a known amount of deoxyribonuclease I to form a complex of vitamin D-binding protein-monomer-type actin- deoxyribonuclease I. Further, deoxyribonucleic acid is added to the resultant complex to effect its reaction with residual deoxyribonuclease and the mononucleotide and/or oligonucleotide formed are determined.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a novel and useful method for quantifying vitamin D-binding proteins present in mammalian body fluids.

(Conventional Technique) In 1976, Bouillon et al. isolated and purified from human serum and rat serum a protein that shows an extremely high affinity for vitamin D, especially vitamin Ds and 25-hydroxyvitamin D (Hur, J. Biochem. ,
66285-291 (19'76)). This protein is called vitamin D binding protein.
ing ProteirB (hereinafter referred to as DBP). Bouillon et al. immunized domestic rabbits with this to obtain the antibody, and used this to quantify DBP contained in serum by immunodiffusion method. As a result, DBP was found to be present in serum at 13 μh in male rats and 9 μh in female rats.
In humans, it was found to exist at a concentration of 330 μB in 7 ml.

5chultze in the 1960s)1. E. et al. as one of the glycoproteins in the α-globulinoid region in human serum.

Consists of 434 amino acids and has a molecular weight of 51,000
A group specific component (Gc) was found. Regarding Gc, it is known that there are mainly three types of Gc that differ in shape and mobility in immunoelectrophoresis. Other than the three types of Gc groups, special forms of Gc have also been discovered. These Gcs only differ in electrophoresis, but their molecular weights and properties remain the same. It is 198 that this Gc is exactly the same substance as the above DBP.
It was revealed in 0.

The Journal of Biological
Chemistry 255.

2270-2272 (1980), DBP, that is, Gc, has the property of binding to actin at a molar ratio of 1:1.

It has been shown that this actin corresponds to the cytoplasmic 4S fraction.

In addition to being quantified by the immunological method of Bouillon et al., DBP is also determined by utilizing its affinity with the vitamin D.

It can also be quantified by reacting tritium-labeled vitamin D with DBP and measuring the radioactivity of tritium incorporated into DBP. The former method is complicated to operate, and the latter method lacks versatility because it deals with radioactive materials. In any case.

In conventional methods for quantifying DBP, there is no concept of utilizing the affinity between DB, P, and actin.

(Objective of the Invention) An object of the present invention is to provide a method for simply and accurately quantifying DBP present in mammalian body fluids such as serum and lymph fluid.

(Structure of the Invention) The method of the present invention was completed based on the inventors' knowledge that DBP forms a complex with actin at a molar ratio of 1:1, and this complex also has the ability to bind to deoxyribonuclease I. Therefore, the method for quantifying vitamin D-binding protein of the present invention consists of (1) adding excess monomeric actin to a mammal-derived sample solution containing vitamin D-binding protein; a step of forming a D-binding protein-monomer actin complex, (2) a step of converting excess monomer actin in the reaction system to polymer actin, (3) a reaction comprising the complex and polymer actin. a step of adding a known amount of deoxyribonuclease I to the system to form a vitamin D binding protein-monomer actin-deoxyribonuclease I complex; (4) adding deoxyribonucleic acid to the reaction system and adding this to the remaining deoxyribonuclease; (5) measuring the mononucleotide and/or oligonucleotide produced by the enzymatic reaction, thereby achieving the above object. - In the method of the present invention, first, excess monomeric actin is added to a sample solution containing DBP. There are two types of actin: monomeric actin (G-actin) and polymeric actin (F-actin), and an equilibrium relationship exists between the two in cells. This G-actin binds to DBP to form a DBP-G-actin complex with a 1:1 molar ratio. In serum, DBP is a few percent of that amount.
(about 4%) exists in combination with vitamin D.

This does not affect complex formation with G-actin. This is because the binding site of DBP with vitamin D is different from the binding site with G-actin. As a result, the above reaction system contains the DBP-G actin complex and excess G actin. Next.

This excess G-actin is converted to F-actin.

For this conversion, for example, 2 the temperature of the reaction system is increased; the IPN is changed to increase the ionic strength of actin; and an actin polymerization accelerator such as adenosine triphosphate, magnesium chloride, phalloidin, etc. is added. There are other methods. However, once actin binds to DBP, it is not converted to F-actin. A known amount of deoxyribonuclease 1 is then added to this reaction system. Deoxyribonuclease (2) also has the ability to bind to the DBP-G actin complex. Deoxyribonuclease I also has the ability to bind to G-actin, but G-actin is not present in the reaction system, and all of the initial surplus G-actin is converted to F-actin. Since deoxyribonuclease I has almost no binding ability to F-actin, deoxyribonuclease I
binds to the DBP-G actin complex in a 1:1 molar ratio. Since its binding site is also the enzymatic active site of deoxyribonuclease I, the formed DBP-G actin-deoxyribonuclease I complex has no enzymatic activity toward deoxyribonucleic acid (DNA). When DNA is added to this reaction system, the remaining deoxyribonuclease I that has formed the DBP-G actin-deoxyribonuclease 1 complex reacts with it. DNA
is decomposed to produce mononucleotides and/or oligonucleotides. This product was converted into 260nWl
quantified by measuring the increase in absorbance with a spectrophotometer. If you perform the above operation using several types of DBP solutions with known concentrations and create a calibration curve in which the read absorbance corresponds to each DBP concentration, you can know the DBP concentration by reading the increase in absorbance. be able to.

Actin used in the method of the present invention is known to exist in animal muscle cells and is involved in muscle movement. In recent years, it has become clear that actin also exists in non-muscle cells, and many proteins that regulate actin have also been discovered. and,
Formation of cytoskeleton and cell division through the action of actin and its regulatory proteins.

It is becoming clear that cell movement9 secretion, phagocytosis, adhesion, proliferation, etc. are controlled. From this situation,
When a cell lesion 9, such as canceration, occurs, it is thought that this affects actin in some way. Toh et al. induced liver cancer in rats using 3°-methyldimethylaminoazobenzene (3′-methylDAB) and performed fluorescence histochemical observation of the liver cells to identify precancerous liver cells and reported that actin-like contractile protein is increased in liver cancer cells (Brlt.

J, Cancer 135+ 761 (1977))
e Vandekerckhove.

Abnormal actin molecules have been discovered in transformed cells derived from human normal diploid cells (Ce1l, 22゜89
3 (1980)). This abnormal actin is human normal β-
Although it is similar to actin, the 244th amino acid out of the 374 amino acid sequence is changed by a point mutation from glycine to aspartic acid. In this way, actin is thought to have some relationship with intracellular malignancy. Understanding changes in actin within cells could provide clues to changes such as cell canceration. However, since actin exists within cells but not in body fluids such as serum, it is impossible to determine the increase or decrease in actin or morphological changes unless cell tissue is collected.

On the other hand, DBP is present in body fluids of mammals, especially in serum.
It is present in extremely high concentrations of 0-500 μg 7 ml. A few percent of DBP in serum binds to vitamin D and is involved in the transport of vitamin D, but the remaining 90%
Its function and physiological role are still unclear. In 1982, Imawari et al. J, Biologica
l Chen, 257, '8153 (1982
) reported that DBP is synthesized in the liver and secreted into the serum. Since DBP has the property of binding to actin, it is inferred that the function or dysfunction of actin in the liver is reflected in DBP. It is also possible that DBP functions as an actin regulatory protein. Furthermore, if changes in the distribution or structure of actin occur due to malignant transformation of cells within the liver, DBP in serum may be affected in some way. For example, it has been reported that DBP in the serum of patients with liver cirrhosis is much lower than in normal conditions.

In addition, in chronic active hepatitis and ruboid hepatitis, which are considered to be disorders associated with abnormalities in the immune system in the liver, anti-actin antibodies, anti-smooth muscle antibodies, anti-actin antibodies, anti-SP antibodies, and anti-LMA antibodies are present in the blood. is known to specifically increase.

Such an increase in blood concentration of actin antibodies and antibodies of substances present in the liver is considered to be the result of actin and substances in the liver leaking into the blood due to damage to liver cells. Naturally, it is expected that the concentration of DBP in serum will change in such cases as well.

Therefore, measuring DBP in relation to actin according to the present invention provides clues to understand changes in actin present within cells.

It can also be used as a guide for determining the presence or absence of liver disease. DBP' instead of measuring the trace amount of vitamin D present in serum
Since vitamin D can be indirectly quantified by measuring , it can also be used as an indicator to determine whether vitamin D metabolism is occurring normally. The DBP quantification method of the present invention is thus extremely useful medically.

(Example) The present invention will be explained below with reference to examples.

Adenosine triphosphate (ATI'), dithiothreitol (
DTT) and calcium chloride were added and dissolved at a concentration of 0.2 mM, and this was used as a G solution.

G-actin obtained from rabbit muscle in 900 μl of G solution
pg-tl-dissolved. This is 100+'g/m
l! (7) 50 μl of DBP solution was added. Separately, add 2M each of calcium chloride and magnesium chloride to G solution.
It was dissolved to a concentration of 100mM and used as a solution F. Add 50 μβ of solution F to the reaction solution containing the above DBP solution and add 21
Incubate for 60 minutes at °C. Mix this well and
Add 20 μl of deoxyribonuclease I solution to that 20 μl.
1 was added thereto, and the mixture was allowed to react at 4°C for 1 minute. Deoxyribonuclease I solution was prepared by adding deoxyribonuclease ■ (EC3,1,
21,1), phenylmethylsulfonyl fluoride (PMSF) and calcium chloride
,1mg/mc 10. trM was added to a concentration of 0.1 mM (7). Add more to this solution.

1 ml of deoxyribonuclease (DNA) solution was added and shaken well. The absorbance was measured at 260 rv 2 minutes after the addition of the DNA solution, and the increase in absorbance was read. The DNA solution contains 50 μg/m7 of DNA, magnesium sulfate, and calcium chloride in 1501 Tris-HCl buffer with pH 7.5! , 4mM and 18m
It was prepared by adding it to the concentration of M.

The concentration of DBP solution was 150ttg/mC500μg/m
j! .

The absorbance at 260 nm was measured at 250 μg/nu, Opg/mp through the same operation as above. As shown in the figure, a calibration curve was created with the concentration of DBP on the horizontal axis and the increase in absorbance for 2 minutes after adding the DNAI solution on the vertical axis.

A human serum solution was used as a sample instead of the DBP solution, and the same operation as above was performed to measure its absorbance.

The amount of increase in absorbance is 0. '085, it was confirmed from the calibration curve in the figure that the DBP in this serum was 420 μg 7 ml.

(Effects of the Invention) According to the method of the present invention, DBP present in body fluids of mammals can be easily and accurately quantified by utilizing the affinity between DBP and actin. The method of the present invention is not limited to simply quantifying DBP, but also provides clues to the increase and decrease status of actin, which exists in the body and is thought to regulate various cell activities, and thereby serves as an indicator for identifying lesions in the liver. sell. Since vitamin D, which exists in small amounts in serum, is bound to DBP, measuring DBP can be used as a guide to determine whether vitamin D metabolism is occurring normally.

[Brief explanation of drawings]

The figure is a calibration curve showing the relationship between the concentration of DBP and the amount of increase in absorbance in an example of the present invention. that's all

Claims (1)

[Claims] 1. (1) Adding excess monomeric actin to a mammal-derived sample solution containing vitamin D binding protein to form a vitamin D binding protein-monomeric actin complex; (2) ) converting excess monomeric actin in the reaction system into polymeric actin; (3) adding a known amount of deoxyribonuclease I to the reaction system containing the complex and polymeric actin to convert vitamin D-binding protein- a step of forming a monomeric actin-deoxyribonuclease I complex; (4) a step of adding deoxyribonucleic acid to the reaction system and reacting it with the remaining deoxyribonuclease I; and (5) a step of forming a monomeric actin-deoxyribonuclease I complex. A method for quantifying vitamin D binding protein, comprising the step of measuring nucleotides and/or oligonucleotides. 2. The method according to claim 1, wherein the measurement in step (5) is performed by measuring absorbance at 260 nm. 3. The conversion in step (2) is caused by a change in the ionic strength of monomeric actin in the reaction system, a change in the temperature of the reaction system, a change in the pH of the reaction system, and the addition of an actin polymerization promoter to the reaction system. The method according to claim 1, which is performed by employing at least one of the following. 4. The method according to claim 3, wherein the actin polymerization promoter is adenosine triphosphate, magnesium chloride, or phallocin.