Suzuki, Yukio

Uchida, Kei

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A PROTECTIVE EFFECT OF γ-GLOBULIN ON α-GLUCOSIDASES FROM SPINACH SEEDS

Okayama University Scientific Achievement Repository

Ber. Ohara Inst. landw. Biol.， Okayama Univ. 19 : 57~61 (1986) A PROTECTIVE EFFECT OF r -GLOBULIN ON a-GLUCOSIDASES FROM SPINACH SEEDS Kei UCHIDA and Yukio SUZUKI Enzymes are often unstable and lose their activity when diluted， and the loss of activity can be prevented by the addition of serum albumin， SH-reagent and other substances. The activity of many kinds of enzymes， such as s-amylase， s-galactosidase， lipase， acid phosphatase， esterase， lactate dehydrogenase， hexokinase， glycerokinase， cholesterol oxidase， and glutathione reductase has been assayed in the presence of bovine serum albumin1) ， but the mechanism of the protective action of bovine serum albumin has not yet been made clear. During the investigations on the properties of multiple forms of α-glucosidase from spinach seeds， we found that r -globulin protected these α-glucosidases more effectively than serum albumin. This paper describes the effect of human r-globulin on the stability of a-glucosidases from the seeds of Spinacia oleracea L. cv. Viloflay. MA TERIALS AND METHODS Seeds of Spinac臼 oleraceaL. cv. Viloflay obtained from Yamato Plantation Co. Ltd.， Tenri， Nara Prefecture， were pulverized in a Shibata coffee grinder model Ely-3 (Shibata Chemical App. Mfg. Co. Ltd.， Tokyo). The powdered spinach seeds (1.5 kg) were extracted with 6liters of 0.02 M acetate buffer， pH 5.0， containing 10 % sodium chloride at 4 "C under stirring. After 24 hr-stirring， the suspension was filtered with a centrifugal dehydrator to remove the residue. The residue was re-extracted with 2 liters of the same buffer containing 10 % sodium chlor-ide， and then filtered. The combined filtrate was used as the crude extract of α-glucosidases. We purified four forms of α-glucosidase from the crude extracts of matured spinach seeds by a procedure including frac-tionation with ammonium sulfate， column chromatography on CM-cellulose， Bio-Gel P-150， and hydroxyapatite. Figure 1 shows the chro-matography of partially purified spinach α-glucosidase on a column of CM-cellulose. Full details of further purification procedure on these peak Institute for Agricultural and Biological Sciences， Okayama University， Kurashiki Received April 9， 1986. K. Uchida and Y. Suzuki 58 P3 8.5 7.5 6.5 ? ? ? ? ? ??? ? ?2.5 1.5 2.0 1.0 0.5 5.5 ? ?? ? ? ? ??0.5 4.5 3.5 ...:.-町、1.0 1.5 2.0 ??? ? ? ??? ??????????????? ????? ?』??』? ? ?。10 20 30 40 Fraction Number (Fractlon volume: 600ml) 1. Elution patern of a-glucosidases from seeds of SpinaCUl o/eracea L. on 1 stCM-cellulose column chromatography FIG. fractions (Pl， P2， P3 and P 4 : a-glucosidases 1， 1， m and N) will be given elsewhere. Each of these purified enzyme preparations was homogeneous on polyacrylamide disc gel electrophoresis at pH 4.0. The molecular weight markers (non-enzymatic proteins) were obtained from Schwarz Bio Research， Orangeburg， N. Y.， USA; apo・ferritinfrom horse (mw 480，000)， r -globulin from human cohn fraction 1 (mw 160，000)， albumin from bovine serum (mw 67，000)， ovalbumin from chick egg (mw 45，000)， chymotrypsinogen A from bovine pancreas (mw 25，000)， myoglo-bin from horse skeletal muscle (mw 17，800)， and lysozyme from chick egg white (mw 14，307). These markers were used to examine their effects on Effect of r-Globulin on Spinach a-Glucosidases 59 the stability of the enzymes. Maltose (HHH) (Hayashibara Biochemical Laboratories， Inc.， Okayama， Japan)， Triton X・100(Nakarai Chemicals， Ltd.， Kyoto， J apan) and s-galactosidase from Esherichia coli (Boehringer Mannheim GmbH， Mannheim， Germany) were obtained from commercial sources. Other chemicals and reagents used were the same as those de-scribed previously5). The a-glucosidase activity was assayed in a reac-tion mixture containing 0.25 ml of 0.1 M acetate buffer at pH 5.0， 0.1 ml of enzyme solution， 0.1 ml of 2 % maltose aqueous solution and distilled water to a final volume of 0.5 ml， and incubation at 37 t for 30 min. Af-ter incubation， the reaction was stopped by heating the mixture in a boil-ing water bath for 10 min. The amount of glucose formed was determined by-the method of Papadopoulos and Hess4) ， asmodified by Dahlqvist2) •One unit of enzyme activity was defined as the amount of enzyme that produced 1μmole/min of glucose from maltose in the absence of marker. RESUL TS AND DISCUSSION During the examination on the properties of multiple forms of α-glucosidases from spinach seeds， these enzymes were found to be inst-TABLE 1. Effects of molecular weight markers， Triton X・1∞and2-mercaptoethanol on activity of spinach a-glucosidase 1. Exp. No. Additions None Apo-ferritin r-Globulin Albumin Ovalbumin Chymotripsinogen A Myoglobin Lysozyme Triton X-I00 2-Mercaptoethanol I H Glucose formed (μmoles/0.5ml of reaction mixture) 0.367 0.257 0.421 0.406 0.535 0.525 0.503 0.484 0.472 0.457 0.469 0.457 0.457 0.435 0.447 0.421 0.360 0.252 0.358 0.250 Exp. 1. The reaction mixture (0.5 ml) containing 0.25 ml of O.IM acetate buffer， pH 5.0，0.05 ml of either 0.5 % marker， 0.5 % Triton X-I00， 10mM 2・mercaptoehtanolor distilled water， 0.1 ml of enzyme solution (0.012 unit) diluted 100 times with distilled water just before use， and 0.1 ml of 2 % maltose aqueous solution was incubated at 37t for 30 min. Exp. 1. The enzyme (O.lml， 0.012 unit)， immediately after dilution， was preincubated at 0 t for 3 hr with 0.25 ml of O.IM acetate buffer， pH 5.0. in the presence or absence of 0.05 ml of the addition. Then， the activity remaining was assayed after the addi tion of 0.1 ml of 2 % maltose aqueous solution. 60 K. Uchida and Y. Suzuki able. The purified preparations suspended in ammonium sulfate solution were stable after storage for several months in a refrigerator， but were un-stable when diluted. As shown in Table 1，α-glucosidase n solution consisting of a 400 times dilution lost about 30 % of its activity in 3 hr at o t. This loss of activity was prevented by the addition of either of a marker. The relative rates of hydrolysis of maltose in the presence of a marker was higher than that in its absence. The effect of )'-globulin was the most remarkable， and observed even at the low concentration of 25 μg per 0.5 ml of reaction mixture. None of the markers tested had any effect on the determination of glucose by glucose oxidase. The addition of 2・mercaptoethanol，a useful SH-protective reagent， was ineffective in preventing the loss of activity. Mat~ui et a1.3) rep~rted that the detergent， Triton X・1∞， contributed to the stability of sweet corn a-glucosidase， butwe found no effect of Triton X・100on spinach α-glucosidases. Both )'-globulin and albumin were effective in preventing the loss of the acti-vities of a-glucosidases 1， m， and N in either incubation at 37 t or preincubation at 0 t and 37 t (Table 2). These results suggest that the markers tested here do not participate in the activation of the α-glucosidase n， but prevent the loss of the enzyme activity during prein-cubation and incubation after dilution. r-Globulin protected al four en-zymes much more effectively than albumin or other markers. The protec・T ABLE 2. Effects of albumin and y.globulin on activities of spinach a-glucosidases. Exp. No. I E ??骨Glucosidases Additions Glucose formed Umoles/0.5ml of reaction mixture) None 0.467 0.345 0.355 1 Albumin 0.591 0.566 0.576 ".Globulin 0.6ω0.637 0.637 None 0.506 0.453 0.406 皿 Albumin 0.668 0.659 0.651 y-Globulin 0.769 0.755 0.753 None 0.359 0.269 0.252 N Albumin 0.458 0.417 0.382 子Globulin 0.547 0.515 0.496 Exp. 1: The assay conditions were the same as described in the Exp. 1 in Table 1， except that human y.globulin and bovine serum albumin were used. Enzymes used were 0.016 (1)，0.017 (皿)， and 0.012 units (N). Exp. n: The assay conditions were the.same as described in the Exp. 1， except that the preincuba. tion was carried out at 0 t:for 20 hr. Exp. m: The assay conditions were the same as described in Exp. n， except that the preincubation was carried out at 37t: for 1 hr. Effect of r.Globulin on Spinach骨Glucosidases 61 tive effect of human r-globulin was also observed to be 1. 4-2.2 times in the assay of s-galactosidase from Eshe吋chiacoli， using the reaction mix-ture (1. 5 ml) containing 0.4 ml of 0.2 M phosphate buffer， pH 7.2， 0.1 ml of enzyme solution (0.15 unit)， 0.5 ml of 1. 2 % 0・nitrophenyl-s -galactopyranoside， 0.15 ml of 0.5 % h uman r-globulin and distilled wa-ter. The mechanism of the protective effect of r-globulin is now under in-veshgahon. SUMMARY r-Globulin protected al four α-glucosidases from the matured seeds of Stinacia oleracea L. cv. Viloflay much more effectively than albumin and other non-enzymatic proteins. r-Globulin was also a potent protector of βgalactosidase from Escherichia coli. REFERENCES 1) Biochemicals diagnostics 1982ー 1983catalogue from Boehringer Mannheim Yamanouchi Co. Ltd.， Tokyo. 2) Dahlqvist， A. 1961. Determination of maltase and isomaltase activities with a glucose-oxidase reagent. Biochem. J.， 80 : 547-551. 3) Matui， H.， Yazawa， 1.and Chiba， S. 1981. Purification and substrate specificity of sweet corn a-glucosidase. Agric. Biol. Chem.， 45 : 887-894. 4) Papadopoulos， N. M. and Hess， W. C. 1960. Determination of neuraminic (sialic) acid， glucose， and fructose in spinal fluid. Arch. Biochem. Biophys.， 88 : 167一171.5) Uchida， K. and Suzuki， Y. 1974. Purification and properties of riboflavin a-glucoside-synthesizing enzyme (a-glucosidase) from pig liver. Agric Biol. Chem.， 38 : 195-206. 

A PROTECTIVE EFFECT OF γ-GLOBULIN ON α-GLUCOSIDASES FROM SPINACH SEEDS

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