JPS60186297A - Removal of nad+ activation inhibitor - Google Patents

Abstract

PURPOSE:NAD<+> containing substance inhibiting NAD<+> activity is brought into contact with NAD<+>-dependent enzymes in a buffer solution to remove the substance, whereby high-purity and high-activity NAD<+> is obtained which can be used in clinical assays. CONSTITUTION:In the preparation of nicotin amide-adenine dinucleotide (NAD<+>) the NAD<+> powder containing NAD<+> activity inhibitors is dissolved in a phosphate buffer to adjust its pH to 6.0. Then, an NAD<+>-depending enzyme (lactic acid dehydrogenase) is added to effect reactions and the reaction mixture is subjected to ultrafiltration to remove the NAD<+>-depending enzyme to give the objective high-activity NAD<+> free from inhibitors.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing highly active nicotinamide adenine dinucleotide (NAI).

More specifically, the present invention relates to a method for producing highly active NAD+ gold by removing NAD+ activity inhibitors mixed in NAI)+.

In general, NAD+ is the coenzyme most widely used today in the fields of biochemical research and clinical testing25, and it is desired to have high purity and high activity.

However, even in NAD+ preparations (7), a decrease in activity is often observed.

A detailed study of NAD+ preparations with reduced activity reveals substances that inhibit NAD+ activity.

Even if we try to study NAD+ activity inhibitors, the structure and properties of the inhibitors are currently completely unknown because separation and purification is extremely difficult.

However, if NAD+ activity inhibitors are contained in NAD+ preparations, they must be removed, as this will have a significant impact on the accuracy of enzyme activity measurements and enzyme reaction analysis using NAD+ preparations. be. NA
Since the physical properties of the D10 activity inhibitor are unknown,
At present, we do not even have a clue as to how to separate this substance.

The present inventors conducted extensive research to remove NAD ÷ activity inhibitor from NAI) specimens, and found that NAD'' activity inhibitor was adsorbed to NAD-dependent enzyme, and NAD +
I knew everything about being separated from the world.

The present invention provides NAD + containing an NAD + activity inhibitor.
and one or more types of NAD4°-dependent enzymes in a buffer solution, the amount of N contained in NAI)+ is reduced.
It is illegal to remove NAD + activity inhibitors, which is characterized by removing activity inhibitors.

In the present invention, NAD+ dependent enzymes include lactate dehydrogenase (LDH) and alcohol dehydrogenase (A I) H
), malate dehydrogenase (MDH), sorbitol dehydrogenase (S D [(), glutamate dehydrogenase (ol
Dtl), etc., but LDH is most preferred.

According to the present invention, N containing an NAD activity inhibitor
When the AJ) + preparation is allowed to coexist with LDH etc. in an appropriate buffer solution, the inhibitory substance is rapidly adsorbed to LDH etc. and the NAJ
) 4-', NAD+ containing no inhibitory substances can be easily prepared. The type of buffer used here may be any buffer as long as it is normally used in enzyme reactions. Specific examples include phosphoric acid, Tris-hydrochloric acid, triethanolamine-hydrochloric acid, histidine-hydrochloric acid, glycylglycine, and Gud buffer. The concentration of the buffer solution may be anywhere from 10mM to 500mM, preferably from 50mM to 208mM.

The strength of the buffer solution is preferably 5.5 to 90, preferably 0 to Z5.

In addition, any type of NAD+ dependent enzyme,
It can be from any source. As sources of LDH, animal sources such as rabbit muscle, pig heart, pig muscle, bovine heart, bovine muscle, leuconostoc, mehinteroides,
Bacterial sources such as high-quality bacteria (II, plant sources, etc., but I, D) from any source can be used.Also, the enzyme used can be in either solution or immobilized form. However, the immobilized state is more preferable for recovering the NAD+ specimen.

Enzyme immobilization methods include the co-binding method, ion binding method, physical absorption method, cross-linking method, and entrapment method.On the other hand, immobilization carriers include polysaccharides such as hillulose, agarose, and dextran. The porosity may be cis, polyacrylamide, carrageenan, polyurethane, etc., but any method may be used, and 1° may be used.

NAIL' and NAI)+-dependent enzymes are brought into contact in a buffer solution, and the contact reaction temperature may be anywhere from 2°C to 40°C, preferably at 2°C to 10°C.

Through this treatment, NAD'-activity inhibitors are adsorbed onto the enzyme, so if the enzyme is separated, highly active NAI)+?
r- can be obtained.

If the enzyme is immobilized, drain the entire column with a buffer solution that retains the NAD+ sample (7.
AI) + activity inhibitors can be adsorbed onto enzymes to create a variety of products.

Next, examples of the present invention will be shown.

Taishi I111゜10 (] +nM IJ 7 acid buffer (p) 16.5
) to dissolve about 1 NAD+ powder [JO//l17'i and 4N
After adjusting the pH to 6.0 with KOH, add it for about 10 minutes.
Add 000 units of I and D H and react at 10°C for 10 minutes! :Ta.

The entire reaction solution was subjected to ultraviolet F, the F solution was collected, and LDH was removed.
'cNAD' was collected. The analysis results for the collected NAI)+ solution are shown in Table 1.

Incidentally, the degree of inclusion of the inhibitory substance in NAD+ is measured as follows.

0,1 MTris-[(Cl(pH8,5) 0.9
0m1. , 80mM NAD 4-solution (pH 8,5)
Approximately 8 u/ml rabbit muscle LDH was placed in a gold cube and incubated at 25° C. for 5 minutes. then 4
Add 12 d of 0 mM L-lactic acid and add 2 d using a spectrophotometer.
Absorbance change per minute at 5°C is measured by fc measurement.

ΔAO7/min for the standard NAn+ that does not seem to contain inhibitors and ΔA for the NAD+ sample
Table 1 Quality of NAD+ when treated with liquid LDH * Eshinkin of LDH used for treatment of NAD+ containing all 1.11M substances show.

*H11 + S2 LDH3001 to 7 frose 30M using Brom/Anne method
0 to a column packed with 3 mJi of n9-fixed LDH gel.
．． 1M K-PO, (p)JlO) Moisturize the gold flow column. Then 100 m9. l ml N
A1. ) + solution (pH”10) flow into 1 ml column (2,
NAD+ was then eluted with the same buffer.

Example 1 Regarding NAI)+ recovered from Carano.
Analyzes were conducted in the same manner. The analysis results are shown in Table 1.

2 Establishment of an okiya 1. l) NAI when treated with H
) + quality fixed T, DH indicates the source of H

Claims (2)

[Claims] (1) NAD+ and N containing NAD+ activity inhibitors
By contacting one or more AD'-dependent enzymes in a buffer solution, the NAD + contained in NAD'° is removed.
A method for removing an NAD+ activity inhibitor, which comprises removing an activity inhibitor. (2) NAD+ according to claim 1, which is characterized in that an NAD+-dependent enzyme is immobilized.
Method for removing activity inhibitors.