• 한국식품과학회지
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• 제34권2호
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• pp.141-150
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• 2002

식품에 함유된 수용성 비타민의 분석을 위하여 식품 중 단백질, 탄수화물 등과 결합하고있는 수용성 비타민을 추출 과정에서 파괴를 최소화하기 위하여 지금까지 통상적으로 행하여온 어떤 화학적 처리도 병용하지 않고 단백질 분해효소인 bromelain과 소의 췌장에서 추출한 protease 및 ${\alpha}-amylase$를 이용하여 결합을 절단하는 순수한 효소처리방법만을 이용하여 추출하였다. 추출된 비타민 시료용액을 $C_{18}$ Sep-Pak solid phase extraction column으로 정제하여 추출된 비타민을 한 column상에서 한번에 모두 역상 HPLC로 분석하는 방법을 개발하였다. 이 방법으로 표준 수용성 비타민의 회수율과 돼지고기 및 감자 중의 수용성 비타민분석 결과와 이 들 식품에 수용성 비타민을 강화하였을 때 강화 비타민의 회수율을 측정한 결과를 A.O.A.C.방법에 따라 추출 정제하여 분석한 결과와 비교하였다. Bromelain 처리와 A.O.A.C.법에서 표준수용성 비타민 중 folic acid가 가장 낮은 37.63%와 48.18%를 나타내었고 protease 처리에서는 pyridoxal이 가장 낮은 75.45%를 나타내었다. Niacinamide를 제외하고는 효소처리법이 A.O.A.C.법보다 높은 회수율을 보였다. 돼지고기 중 수용성비타민은 효소 처리방법에서 aminobenzoic acid, pyridoxal, pyridoxine, niacinamide 및 thiamin 등 6가지가 검출되었고 $C_{18}$ Sep-Pak column에 의한 정제에도 불구하고 불순 peak들이 많이 나왔으나 정량에 방해되는 불순 peak는 없었고 A.O.A.C.방법에 의한 추출 정제 시 niacinamide와 thiamin정량에 있어서 불순 peak에 의하여 정확한 정량이 불가능하였다. 돼지고기에서 특히 단백질과 공유결합을 형성하고 있는 비타민 $B_6$의 경우 문헌에 보고된 값들 보다 상당히 높은 값으로 정량되어 단백질 함량이 높은 시료에 대하여 별도의 정제과정이 추가로 필요한 것으로 나타났다. 비타민 강화 돼지고기 중의 강화 비타민의 회수 율은 표준 비타민 보다 회수율이 높고 정확하였다. 감자의 경우 protease처리에서 aminobezoic acid, pyridoxal, pyridoxine 및 thiamin 등 4가지가 검출되었고 bromelain 처리에서는 pyridoxal이 검출되지 않았다. 효소처리법에서 돼지고기와 마찬가지로 $C_{18}$ Sep-Pak column에 의한 정제로도 불순 peak가 많았으나 정량에 방해되는 불순 peak는 없었다. A.O.A.C.방법에서 pyridoxamine과 thiamin정량에서 불순 peak로 인하여 정확한 정량이 불가능하였다. 비타민 $B_6$의 경우 돼지고기와 달리 문헌에 나타난 값들과 비슷한 결과를 얻었는데 이는 단백질 함량이 낮은 관계로 판단된다. 비타민 강화 감자에서 강화 비타민의 회수 율도 돼지고기와 마찬가지로 상당히 정확하였고 A.O.A.C.법보다 높게나왔다.

• Asian Pacific Journal of Cancer Prevention
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• 제13권11호
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• pp.5333-5338
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• 2012

Vitamin B6 functions as a coenzyme in >140 enzymatic reactions involved in the metabolism of amino acids, carbohydrates, neurotransmitters, and lipids. It comprises a group of three related 3-hydroxy-2-methyl-pyrimidine derivatives: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphorylated derivatives [pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP)], In the folate metabolism pathway, PLP is a cofactor for the mitochondrial and cytoplasmic isozymes of serine hydroxymethyltransferase (SHMT2 and SHMT1), the P-protein of the glycine cleavage system, cystathionine ${\beta}$-synthase (CBS) and ${\gamma}$-cystathionase, and betaine hydroxymethyltransferase (BHMT), all of which contribute to homocysteine metabolism either through folate-mediated one-carbon metabolism or the transsulfuration pathway. Folate cofactors carry and chemically activate single carbons for the synthesis of purines, thymidylate and methionine. So the evidence indicates that vitamin B6 plays an important role in maintenance of the genome, epigenetic stability and homocysteine metabolism. This article focuses on studies of strand breaks, micronuclei, or chromosomal aberrations regarding protective effects of vitamin B6, and probes whether it is folate-mediated one-carbon metabolism or the transsulfuration pathway for vitamin B6 which plays critical roles in prevention of cancer and cardiovascular disease.

• BMB Reports
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• 제29권4호
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• pp.321-326
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• 1996

Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with $NaBH_4$. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.

• Animal cells and systems
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• 제10권4호
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• pp.197-201
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• 2006

Diaminopimelate decarboxylase (DAPDC, EC 4.1.1.20) catalyzes the conversion of diaminopimelate into lysine (Lys), which is the last step in Lys biosynthetic pathway. The genes for DAPDC have been reported in many bacteria, and more recently in Arabidopsis. Here we report characterization of a gene for DAPDC from rice (OsDAPDC). Sequence analysis of a cDNA clone revealed a full-length open reading frame for OsDAPDC that encoded 490 amino acids, approximately 53.2 kDa protein. The OsDAPDC protein contains a consensus binding site for pyridoxal-5'-phosphate as a cofactor and has a sequence at the amino terminus that resembles a transit peptide for localization to plastids, similar to that of Arabidopsis. Single gene encoding DAPDC was found in chromosome II in rice. The predicted amino acid sequence of OsDAPDC is highly homologous to that of the enzymes for DAPDC encoded by lysA of many bacteria. Expression of OsDAPDC in lysA mutants of Escherichia coli shows that the gene is able to functionally complement the mutants. These results suggest that OsDAPDC encodes a protein for diaminopimelate decarboxylase in rice.

• Journal of Nutrition and Health
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• 제30권4호
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• pp.425-433
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• 1997

Levels and distribution of five B-6 vitamers(PMP, PM, PLP, PL, and PN) and pyridoxine $\beta$-glucoside conjugates(PN-glucoside) were examined in milk of American women who received supplements of 2.5 or 10mg PN HCl/d and of unsupplemented Egyptian women during the first six months of lactation. B-6 vitamer and PN-glucoside levels in human milk were determined by reverse-phase HPLC. Pyridoxal(PL), which has been reported to be the most rapidly absorbed form of vitamin B-6 and may facilitate bioavailability, was the predominant vitamer in human milk of all three groups. Pyridoxal made up 72% of total vitamin B-6 for the 2.5mg supplemented group, 76% for the 10mg group, and 59% for the Egyptian group. Level and Percent PL were significantly lower for Egyptian women. Mean growth of the two American groups was similar to each other and within the normal range of the NCHS reference, however, Egyptian infants showed growth faltering at 6 months. The Percent of PN-glucoside, a less bioavailable form of vitamin B-6 in humans was 1% in milk of American women and was 11% in Egyptian women and these values were significantly different. for Egyptian women, total vitamin B-6 levels in breast milk correlated Positively with animal protein intake(r=0.91) and percent PN-glucosides(r=0.53) and negatively with plant protein intake(r=-0.55). These findings showed that high plant protein intake was associated with low concentrations of PL and total vitamin B-6 in human milk.

• Nutritional Sciences
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• 제5권1호
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• pp.20-25
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• 2002

The vitamin $B_{6}$ status of 49 healthy college student (women, aged 20-26 y) was estimated for evaluation of vitamin $B_{6}$ status and the Korean Recommended Dietary Allowance (RDA) for vitamin $B_{6}$. The average daily vitamin $B_{6}$ intake of the subjects was 0.86 $\pm$ 0.289 mg/d or 61.43 $\pm$ 24.10% of Korean RDA. The average ratio of vitamin $B_{6}$ intake to daily protein intake was 0.014 $\pm$ 0.003 mg/g protein. Foods from animal and plaint sources provided 34.25 $\pm$ 18.62% and 65.78 $\pm$ 18.72%, respectively, of total vitamin $B_{6}$. Plasma pyridoxal 5'-phosphate (PLP) concentration was significantly (p<.01 - p<.001) positively correlated to intakes of all other nutrients except vitamin C. However, no significant correlation was found between plasma PLP and nutrient intake. Vitamin $B_{6}$ intake only tended to have a positive correlation with plasma PLP concentration. Plasma total cholesterol was correlated to plasma PLP concentration (p<.05). Plasma PLP had no correlation with levels of glucose, triglyceride, and albumin. These results confirm that the present Korea RDA for vitamin $B_{6}$ of 1.4mg/d based on 0.02 mg/g protein is adequate.

• BMB Reports
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• 제38권1호
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• pp.58-64
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• 2005

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

• Preventive Nutrition and Food Science
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• 제14권4호
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• pp.358-361
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• 2009

Aldose reductase was purified to electrophoretic homogeneity from porcine liver. The purified enzyme was a monomer of 36 kDa. The enzyme was strongly inhibited by $Cu^{2+}\;and\;Mg^{2+}$ ions. Incubation of the enzyme with pyridoxal 5'-phosphate led to complete inhibition of enzymatic activity, suggesting that lysine residue is involved at or near the active site of the enzyme. The enzyme exhibited a broad substrate specificity. Furthermore, the enzyme was capable of decolorizing Alizarin, an anthraquinone dye.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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• pp.270-270
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• 1994

돼지 뇌조직으로부터 순수 분리 정제된 Glutamate decarboxylase (GAD)는 효소 dimer당 0.8mole 보조 인자인 pyridoxal-5-phosphate(PLP)가 강하게 binding되어 있었다. 이러한 부분적으로 resolved된 효소에 외부로부터 PLP를 넣어주면 효소의 활성도는 최대값으로 증가하였다. 정제된 GAD는 sulfydryl시약에 의한 화학변형에 의하여 효소의 활성도를 상실하였으며 환원제인 dithiothreitol이나 2-mercaptoethanol의 첨가에 의하여 효소의 활성도가 복구되는 것으로 보아 효소의 활성부위의 활성에 직접 관여하는 중요한 cysteinyl잔기가 존재하고 있는 것을 알 수 있다.

• 약학회지
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• 제22권1호
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• pp.15-21
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• 1978

Gentamicin sulfate reacted with pyridoxal and zinc (II) ion in pyridine-methanol solution to yield highly fluorescent zinc(II) chelates of N-pyridoxylidene derivatives. This fluorescence reaction was sensitive and showed excitation maximum at 398nm, and emission maximum at 482nm. The effects of reagent concentration, reaction time and temperature, standing time and temperature were studied. And a new fluorophotometric method for the determination of gentamicin sulfate was developed. A good result was obtained and this method was applied to various preparations.