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    • PPM-18 receptor In this study using pure enzyme preparations

    2023-01-30

    In this study, using pure enzyme preparations of human placental recombinant AR (hAR) and of AR isolated from bovine lens (bAR), which behave as Michaelis PPM-18 receptor with glyceraldehyde as substrate, is shown that an apparent negative cooperativity action for glucose still occurs. We present evidence for a partial inhibitory action of the hemiacetal form of aldoses on the AR activity. A kinetic model is proposed to explain the apparent negative cooperative behavior observed for glucose and more generally for aldoses that are able to produce cyclic hemiacetal structures.
    Materials and methods
    Results
    Discussion The biphasic kinetic behavior occasionally reported for the reduction of different substrates, including d-glucose, by AR and associated with the possible presence of two enzyme forms [16], [18], [21], [25], [26], was also observed in the present work, in which all precautions were taken to prevent an oxidative modification of the purified bAR and hAR. Thus, DTT, as thiol reducing agent, and EDTA (also present in the AR assay mixture) never left AR during extraction and purification (except, necessarily, for the time needed to perform the affinity chromatography step). In addition, the use of 2-ME was completely avoided. In fact, 2-ME, so often used as a preservative for reduced thiols, has been found to be a very efficient trigger of AR oxidative modification, when often uncontrolled factors (storage time and conditions, temperature, traces of transition metal ions, disulfides) induce its full or partial oxidation [29]. Paradigmatic is the reported evidence of a purified AR preparation that was isolated from human psoas muscle in the presence of 5mM 2ME and which resulted to be inactivated (we would say re-converted to its native form) by DTT, but not by 2ME [48]. Finally, the ionic strength stress, as occurs during ammonium sulfate fractionation, which forces the release from AR of the bound pyridine cofactor [39], especially during earlier purification steps, was avoided in order to preserve AR in its native state [30], [49]. With the above-mentioned precautions, the final highly-purified preparations of both bAR (data not shown) and hAR behave, as expected, as monomeric catalysts characterized by simple hyperbolic kinetics when d-erythrose, l-threose, GAL, HNE and 4-nitrobenzaldehyde were used as substrates in a range of concentrations in which substrate inhibition phenomena can be ruled out (Fig. 2). The linearity observed with GAL, l-threose or d-erythrose by itself does not rule out the presence of two enzyme forms. In fact an inhibitory adduct between the enol form of the substrate and the oxidized pyridine cofactor, which would mask the biphasic behavior expected as a consequence of the presence of two enzyme forms, can be generated [27]. However, the linearity observed with HNE, which is more susceptible as a target by nucleophilic attack at the C3 than as nucleophile on the pyridine ring of NADP+, and with 4-nitrobenzaldehyde, a molecule that is unable to enolize and therefore is not able to generate the inhibitory adduct, confirms the absence of different forms of AR in the used enzyme preparations. The results obtained with d-glucose (Fig. 1) enable two couples of kinetic parameters to be estimated, namely and , measured at high and at low substrate concentrations. For each kinetic parameter an approximately 10 fold difference was observed between the high and low ranges of substrate concentration. These results explain the wide range of kinetic parameters values reported to date for glucose in the literature [30], [50], [51], [52], [53], [54]. However, having ruled out the presence of two enzymatic forms concurring to the reduction of glucose, as is the case of the present study, interpreting the above apparent kinetic parameters becomes conceptually difficult. Thus, the observed apparent negative cooperative reduction of d-glucose was associated with the particular features of this substrate, rather than with the combined action of two different enzyme forms. We found that this effect was the result of a partial inhibitory action exerted by the glucose hemiacetal on the reduction of glucose free aldehyde. This biphasic behavior is not unique for d-glucose, but is a general feature of the long chain aldoses that generate cyclic hemiacetals. In fact, the reduction processes of l-idose, d-galactose, d-ribose and d-xylose catalyzed by hAR, all display apparent negative cooperative features (Fig. 3). It thus follows that the proposed interaction of cyclic hemiacetal with the enzyme is not a special feature of glucose hemiacetal, but applies to the hemiacetal structures of the tested 5 and 6 carbon atom aldoses, which all modulate the reduction of their respective open aldehyde forms.