临床检验杂志
臨床檢驗雜誌
림상검험잡지
2001年
1期
4-7
,共4页
乳酸脱氢酶%红细胞%提纯%动力学性质
乳痠脫氫酶%紅細胞%提純%動力學性質
유산탈경매%홍세포%제순%동역학성질
目的研制乳酸脱氢酶的标准品，从人红细胞中提取了乳酸脱氢酶(LD)并对其特性进行了研究。方法＃用经过改进的方法，包括完全溶解红细胞，羟磷灰石处理，硫酸铵沉淀，CM-Sephadex、C50、SephadexG100和5'-AMP-Sepharose亲和层析。结果该提制品LD比活性达163.0kU/g蛋白，几乎不含其它杂酶(测不到ALT、AST、CK、GGT、ChE、ALP、Amy活性)。聚丙烯酰胺凝胶电泳(自然凝胶系统)酶染色显示LD1、LD2区带，蛋白质染色显示与酶相应的区带。37℃条件下，正向反应中，对底物L-乳酸和NAD的米氏常数(Km)分别为1.040和0.192mmol/L；逆向反应中，对底物丙酮酸和NADH的Km分别为0.119和0.039mmol/L。结论提纯的LD其催化性质和人混合血清LD非常相似，本研究为今后进一步研制LD测定用参考品建立了基础。
目的研製乳痠脫氫酶的標準品，從人紅細胞中提取瞭乳痠脫氫酶(LD)併對其特性進行瞭研究。方法＃用經過改進的方法，包括完全溶解紅細胞，羥燐灰石處理，硫痠銨沉澱，CM-Sephadex、C50、SephadexG100和5'-AMP-Sepharose親和層析。結果該提製品LD比活性達163.0kU/g蛋白，幾乎不含其它雜酶(測不到ALT、AST、CK、GGT、ChE、ALP、Amy活性)。聚丙烯酰胺凝膠電泳(自然凝膠繫統)酶染色顯示LD1、LD2區帶，蛋白質染色顯示與酶相應的區帶。37℃條件下，正嚮反應中，對底物L-乳痠和NAD的米氏常數(Km)分彆為1.040和0.192mmol/L；逆嚮反應中，對底物丙酮痠和NADH的Km分彆為0.119和0.039mmol/L。結論提純的LD其催化性質和人混閤血清LD非常相似，本研究為今後進一步研製LD測定用參攷品建立瞭基礎。
목적연제유산탈경매적표준품，종인홍세포중제취료유산탈경매(LD)병대기특성진행료연구。방법＃용경과개진적방법，포괄완전용해홍세포，간린회석처리，류산안침정，CM-Sephadex、C50、SephadexG100화5'-AMP-Sepharose친화층석。결과해제제품LD비활성체163.0kU/g단백，궤호불함기타잡매(측불도ALT、AST、CK、GGT、ChE、ALP、Amy활성)。취병희선알응효전영(자연응효계통)매염색현시LD1、LD2구대，단백질염색현시여매상응적구대。37℃조건하，정향반응중，대저물L-유산화NAD적미씨상수(Km)분별위1.040화0.192mmol/L；역향반응중，대저물병동산화NADH적Km분별위0.119화0.039mmol/L。결론제순적LD기최화성질화인혼합혈청LD비상상사，본연구위금후진일보연제LD측정용삼고품건립료기출。
Objective To preparing the reference material(RM) of lactate dehydrogenase(LD),we purification of LD from human erythrocyte(RBC)and studied its properties.Methods Using a modified procedure which including complete hemolysis of the RBC,hydroxyapatite treatment,(NH4)2 SO 4 Precipitation,CM- Sephadex C 50,Sephadex G100 and 5'- AMP- Sepharose 4B affinity chromatography.Results The purified LD has a sepecific activity of 163.0 kU/g protein.It is almost free of contaminating enzymes.Two corresponding bands were observed on both PAG plates stained for either protein or LD activity after electrophresis had been done.The apparent Michaelis constants were of 1.000 and 0.179 mmol/L for L-lactate and NAD and of 0.119 mmol/L 0.062 mmol/L for pyruvate and NADH respectively.Conclusion The final purified LD was found to be very similar to that in human serum in catalytic properties.It is intended to be a fundament to prepare a RM for the measurement of LD.
