Abstract
Aldehyde reductase (ALR1) and aldose reductase (ALR2) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia PBE 94 and then chromatography on a hydroxylapatite high performance liquid chromatography column. Expression of ALR1 and ALR2 in placenta is variable with ALR1/ALR2 ratios ranging from 1:4 to 4:1. ALR1 and ALR2 are immunochemically distinct. ALR1 shows broad specificity for aldehydes but does not efficiently catalyze the reduction of glucose due to poor binding (Km = 2.5 M). ALR1 exhibits substrate inhibition with many substrates. ALR2 also shows broad specificity for aldehydes. Although glucose is a poor substrate for ALR2 compared with other substrates, the affinity of ALR2 for glucose (Km = 70 mM) suggests that glucose can be a substrate under hyperglycemic conditions. ALR2 shows normal hyperbolic kinetics with most substrates except with glyceraldehyde, which exhibits substrate activation. Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde. Dithiothreitol treatment of ALR2 did not alter its properties toward other substrates or affect its inhibition by aldose reductase inhibitors such as sorbinil (2,4-dihydro-6-fluorospiro-[4H-1-benzopyran-4,4'-imidazolidine]-2' ,5'- dione), tolrestat (N-[[6-methoxy-5-(trifluoromethyl)-1-naphthalenyl]thioxomethyl]-N- methylglycine), or statil (3-[(4-bromo-2-fluorophenyl)methyl]-3,4-dihydro-4-oxo-1-phthalazineac etic acid).
Highlights
Aldehyde reductase (ALRl) and aldose reductase (ALRS) were purified from human placenta by a rapid and efficient scheme that included rapid extraction of both reductases from 100,000 x g supernatant material with Red Sepharose followed by purification by chromatofocusing on Pharmacia
Treatment of ALR2 with dithiothreitol converted it into a form that exhibited hyperbolic kinetics with glyceraldehyde
We report the development of a scheme for the rapid isolation of ALRl and ALR2 from human placenta
Summary
6.0 reductase, and ALR3 [8, 10] In spite of these different names, all of these enzymes are capable of catalyzing the reduction of a wide range of aldehydes and ketones, some of which are xenobiotics and others of which are endogenous compounds. The fact that inhibitors of ALR2 can prevent a number of diabetic complications in animal models of diabetes strongly supports the conclusion that the polyol pathway plays a role [15]. Inhibition of glucose reduction through the use of inhibitors of ALR2 (usually called aldose reductase inhibitors) represents a possible method for prevention of diabetic complications in man. It would be preferable to use human ALR2 for these studies because of marked species differences in the aldose reductase inhibitor-binding properties of ALR2. The substrate specificities and drug-binding properties of ALRl and ALRP are compared
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