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InChI
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InChI=1S/C12H22O11/c13-1-3-5(15)6(16)9(19)12(22-3)23-10-4(2-14)21-11(20)8(18)7(10)17/h3-20H,1-2H2/t3-,4-,5-,6+,7-,8-,9-,10-,11-,12-/m1/s1
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激酶实验
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Compatible solute assays for SspI, G6PDH, and ADH were repeated three times with the exception of the G6PDH time course experiment that was repeated twice. SspI is a bacterial restriction enzyme that cleaves double stranded DNA. In this assay, it cuts the pGEX-4T-2 circular plasmid twice and generates 3.77-kb and 1.19-kb bands in the reaction buffer (50 mM NaCl, 100 mM Tris-HCL, 10 mM MgCl2, 0.025% Triton X-100 pH 7.5 at 25C). Five units of SspI in the reaction buffer in the absence of maltose were incubated at 50C for 15 min where 50% of the enzyme activity was lost. Aliquots were incubated at 50C in the presence of 14, 100, 200, and 400 mM maltose. A substrate, 0.5 mg of pGEX-4T-2 circular plasmid, was added to the reaction mixture and incubated at 37C for 1 h. The control reaction did not include maltose and was not exposed to heat treatment to show the full enzyme activity. Twenty-two mL of a 50 mL reaction was loaded on a 1% agarose gel. The gel was run at 100 V for 30 min and stained with ethidium bromide to visualize the product. A 5-kb band representing a single double stranded cut of plasmid was quantified to assess the degree of protection by maltose. Scion Image for Windows was used to quantify the intensity of the ethidium bromide stained DNA bands from the inverse imagesof the gels.The same assay was performed using Suc, trehalose, and Glc to compare the ability of maltose as a compatible solute. A time-course experiment was conducted where SspI was treated in the absence and in the presence of 400 mM maltose, Glc, Suc, and trehalose at 50 C for 0, 5, 10, 15, 20, and 25 min. G6PDH in 50 mM Tris-HCl pH 7.8 was incubated at 48_x005f C for 15 min, which reduced activity by 50%. G6PDH in 50 mM Tris-HCl pH 7.8 was heated in the absence and presence of 14, 100, 200, and 400 mM maltose, trehalose, Glc, and Suc. The control did not include any sugar and was not given a heat treatment. The reaction (1.5 mL) included 2.97 mM MgCl2, 50 mM Tris-HCl pH 7.8, 0.6 mM b-NADP1 (freshly prepared), 10 mM Glc-6-P, and 595 ng heat shocked G6PDH. Production of NADPH was followed for 3 min at A340 using a Lambda 3A UV/VIS spectrophotometer. A time-course experiment was conducted where G6PDH was treated in the absence and in the presence of 400 mM maltose, Glc, Suc, and trehalose at 48C for 0, 5, 10, 15, 20, and 25 min. Lyophilized ADH from yeast 300 U/mg. ADH in 50 mM potassium phosphate buffer pH 7.6 was exposed to 53.5 C for 15 min with a loss of 50% activity. ADH in 50 mM potassium phosphate buffer pH 7.6 was heated in the absence and presence of 14, 100, 200, and 400 mM maltose, trehalose, Glc, and Suc. The control did not include any sugar and was not given a heat treatment. Reaction volume 1.5 mL included 333 mM ethanol, 50 mM potassium phosphate pH 7.6, 4.15 mM b-NAD1 (freshly prepared), and 500 ng heated ADH. Production of NADH was followed for 20 s at A340 using a Lambda 3A UV/VIS spectrophotometer. A time course experiment was conducted where ADH was treated in the absence and in the presence of 400 mM maltose, Glc, Suc, and trehalose at 53.5 C for 0, 5, 10, 15, 20, and 25 min.[1]
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