A study of nad-linked malic enzyme in muscle of the dung beetle catharsius SP. and related genera

Abstract

Proline oxidation appears to constitute a major energy produring pathway in flight and coxal muscle of various dung beetles (Scarabaeidae: sub-family Coprine). This was concluded from the fact that activities likely to be assocIated with a proline energy metabolism such as proline dehydrogenase, alanine aminotransferase and NAD-malic enzyme, were unexpectedly high in both flight and coxal muscle of Coprine beetle, as was found for the better known, proline utilising tsetsefly. In contrast, these enzyme activities were relatively low in various other insects, all claimed to be either lipid and/or carbohydrate utilisers. Major glycolytic enzymes were relatively low in Coprine beetles and the tsetsefly, in contrast to various other known carbohydrate utilising insects, in which these activities were high. This suggests that carbohydrate breakdown might not be important as an energy supplying pathway in this beetle. It is noteworthy that NAD-malic enzyme and alanine aminotransferase were mitochondrial in keeping with a functi6nal role in proline metabolism. Mitochondria from both flight and coxal muscle of Coprine beetles oxidised proline faster than any other substrate tested. This suggests that-proline constitutes a major physiological substrate. In contrast, these mitochondria oxidised pyruvate and lipids at negligible rates. Presumably, beetles are unable to utilise carbohydrates and lipids directly. However; mitochondria from flight muscle of "starved" beetles oxidised glycerophosphate faster than proline and contained higher glycolytic enzyme activities. It is possible therefore that the flight muscle tissue switches to a carbohydrate based metabolism under "starved" conditions. Flight and coxal muscle NAO-malic enzymes were purified using the same procedure and their properties investigated under identical conditions. Specific activities of 28 units/min/mg protein and 25 units/min/mg protein were obtained for flight and coxal muscle enzymes respectively. A similar molecular weight of 250 - 260 x 103 was estimated for the flight and coxal muscle enzymes. The two enzymes had similar electrophoretic and kinetic properties. Catharsius NAD-malic enzyme had complete requirement for Mn2+ or Mg2+ for activity and exhi bited co-operativity wi th respect to both substrates. With malate as the variable substrate, the '. x degree of sigmoidicity and the Krn values increased with pH. Succinate activated the enzyme by increasing the affinity for malate and tended to abolish co-operativity, as well as lowering the Km values. Fumarate al~o activated the enzyme but unlike succinate, high concentrations were strongly inhibitory. The enzyme had relatively high Km values for both substrates, probably as an adoptation to fit it for the control of mitochondrial ma late utilisation. It was maximally 30 - 40% as active w ith NAOP as the NAO-linked activity, and had similar affinity for the coenzymes. With succinate present, linear primary reciprocal initial velocity-substrate plots were obtained. From the patterns of these plots, it was deduced that the Cartharsius enzyme followed a fixed order of substrate binding mechanism with malate binding before NAD. Inhibition patterns 9btained with NADH and bicarbonate were also in keeping with such a mechanism. A Ca2+ dependent OAA decarboxyl ase acti vity was puri fied together with NAD-malic enzyme activity and was probably attributable to the same protein. The reaction catalysed by NAD-malic enzyme was slightly reversible apparently with high pyruvate and bicarbonate and its unlikely that reductive carboxylation has any physiological significance in Corpine beetles. The properties of the Catharsius NAD-malic enzyme were compared with those of otherNAD- and NADP-malic enzymes. The relationship of velocity and malate concentration and the modification by pH, succinate, ATP and metal activator were considered withi n the concept of the monod Wyman Changeux (MWC) model