Purification and characterization of pyruvate carboxylase from the honeybee and some properties of related biotin‐containing proteins in other insects

Abstract

AbstractOur discovery of a Mr 128 k biotin‐containing protein in an extract of the white‐fly was an unexpected outcome of the use of an avidin‐biotin‐peroxidase method to visualize a western blot. This major biotin‐containing protein was shown to be present in several tissues of 10 different species of insects by doing a western blot and staining it with streptavidin‐linked peroxidase. The amount of this protein in the thorax of the mosquito, Aedes aegypti, increased during development. The non‐flying grasshopper, Barytettix psolus, had reduced amounts of this protein in their thoraces compared to a flying grass‐hopper, Schistocerca americana. The major biotin‐containing protein was purified from the thoraces of honeybee, Apis mellifera, using an avidin Sepharose affinity column. The purified biotin‐containing protein was shown to be pyruvate carboxylase, an enzyme that catalyzes the specific transfer of a carboxyl group to pyruvate, yielding oxaloacetate. The purified honeybee pyruvate carboxylase was characterized enzymatically and structurally. This protein had a single subunit of Mr 128 k and formed a native molecule of about Mr 500 k consisting of four of these subunits. The amino acid composition of the protein was also obtained. The enzymatic activity of this protein required acetyl‐CoA, ATP, and Mg2+. The Kms of the enzyme for bicarbonate and pyruvate were similar to pyruvate carboxylase from other oganisms. The biotin‐containing protein was also partially purified from mosquito thoraces using the same methods and was shown to be pyruvate carboxylase. The comparison between insect pyruvate carboxylase and that of other organisms is provided and the possible physiological role of the pyruvate carboxylase in the thoracic muscles of insects is discussed.