E. Melloni

Rabbit liver fructose 1,6-diphosphatase. Properties of the native enzyme and their modification by subtilisin☆

Abstract A simple two-step procedure is described for the purification of fructose 1, 6-diphosphatase from rabbit liver. The enzyme shows activity at neutral pH, in contrast to the previously described “alkaline” fructose diphosphatase. The neutral fructose diphosphatase has a higher molecular weight, 140,000, as compared with 130,000 for the alkaline enzyme and a somewhat different amino acid composition. Most significant is the presence of one residue of tryptophan per subunit; this amino acid is not present in the alkaline fructose diphosphatase. The subunit molecular weight of the neutral enzyme is 36,000, and it contains 4 moles of COOH-terminal alanine. Digestion with subtilisin converts the neutral enzyme to one with properties resembling those of the alkaline form. The pH optimum is shifted to pH 9 and the sensitivity toward inhibition by AMP is decreased. The molecular weight of the modified enzyme is 120,000. The results suggest that the changes in catalytic properties are associated with the removal of a tryptophan-containing peptide or peptides with molecular weights totaling approximately 6000 per subunit, and that the alkaline enzyme previously studied in this and other laboratories is a modified form of the native neutral fructose diphosphatase.

Activation of Ca2+-dependent proteolysis in skeletal muscle and heart in cancer cachexia.

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFα are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca2+-dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca2+-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca2+-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca2+-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca2+-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca2+-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca2+-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia.

Interactions of Zn2+ and Mg2+ with rabbit liver fructose 1,6-bisphosphatase

Abstract Purified rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) binds 12 mol of Zn 2+ /mol, presumably 3 per subunit. Zn 2+ binds to the first set of 4 sites with high affinity and shows positive cooperativity; half-saturation of these sites was at a concentration of 0.03 μ m . The dissociation constant for Zn 2+ binding to the second set of sites is 0.35 μ m . Binding to the third set of sites requires the presence of the substrate, fructose 1,6-bisphosphate, and shows low affinity, with K d ≅ 2.5 μM. Mg 2+ , the activating cation, alters the binding of Zn 2+ to all three sets of sites and together both cations appear to modulate the catalytic activity. When the enzyme is activated with 0.2 m m Mg 2+ , filling of the first set of high-affinity sites by Zn 2+ results in inhibition of catalytic activity; both Zn 2+ binding and inhibition by Zn 2+ are prevented when the concentration of Mg 2+ is raised to 0.1 m , or when EDTA is added. The inhibitory effect of Zn 2+ is also partially reversed by raising the concentration of Zn 2+ and at high concentrations (10 μ m ) Zn 2+ can substitute for Mg 2+ as the activating cation. A tentative model is presented in which the first set of sites is considered to be involved in inhibition by Zn 2+ and the third set in activation by Mg 2+ or Zn 2+ ; the second set of sites of intermediate affinity appears to modulate the catalytic activity between the high and low range, depending on whether these sites are filled by Zn 2+ or by Mg 2+ .

Conversion of "neutral" to "alkaline" fructose 1,6-diphosphatase by controlled digestion with papain.

Abstract When highly purified, homogeneous preparations of “neutral” fructose 1,6-diphosphatase are treated with papain under controlled conditions the enzyme s converted to a form with an alkaline pH optimum. The changes in catalytic properties include modification of the pH profile and changes in the ratio of activities toward fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate. Preliminary data indicate a decrease in the molecular weight of approximately 3000 per subunit.

Binding of monoclonal antibody to cathepsin M located on the external surface of rabbit lysosomes

A monoclonal antibody raised against rabbit liver cathepsin M binds to intact rabbit liver lysosomes. The binding is specific and is abolished by treating the lysosomes with trypsin, which has previously been shown to digest the membrane-bound cathepsin M [S. Pontremoli, E. Melloni, M. Michetti, F. Salamino, B. Sparatore, and B. L. Horecker (1982) Biochem, Biophys. Res. Commun. 106, 903-909]. Rabbit liver lysosomes are adsorbed onto Sepharose 4B coupled to anti-cathepsin M, but not to Sepharose 4B itself or to Sepharose coupled to a nonspecific antibody. The results confirm the location of membrane-bound cathepsin M on the outer surface of the lysosomal membrane.