Brenda E. Ryman

Enzyme entrapment in liposomes

Enzyme replacement therapy for patients with various disorders in which a specific enzyme activity is absent from one or more tissues, has been attempted in several instances by direct administration to the patient of an enzyme designed to remove undesirable accumulation products [I-S] . However, apart from the possible immunological response that could arise from the foreign protein, there are also the problems related to undesirability of having certain enzymes in the circulation and of directing the given protein to a particular tissue. We thought that some of the difficulties mentioned could be circumvented by entrapping proteins into liposomes (lipid spherules). Liposomes are formed when phospholipids are allowed to swell in aqueous media and become hydrated liquid crystals. These, when suitably dispersed, consist of a series of concentric bilayers which alternate with aqueous compartments in which can be entrapped water-soluble substances [6,7]. The present paper describes the entrapment ofAspergiZlus niger amyloglucosidase (E.C.3.2.1.3.) and albumin into liposomes. The choice of the amyloglucosidase in this entrapment investigation reflects our interest in the glycogen storage diseases, while the commercial availability of r3r I-albumin has provided a readily detectable protein, similar in both molecular size and charge to the amyloglucosidase.

The action pattern of amylomaltase.

If the reaction is driven to the right with glucose oxidase, successive transglucosylations lead to the formation of 1 + 4-linked a-glucans that stain blue with iodine. The induction of amylomaltase is accompanied by the formation of a maltose permease [2] and a maltodextrin phosphorylase [3]. The further purification and physicochemical properties of amylomaltase, together with its general properties and mechanism of action have also been documented [4,5]. Our own interest in this enzyme originated primarily in its possibilities as a tool for studying the fine structures of glycogen and amylopectin. In the isolation and assay procedures, however, several unexpected findings prompted further investigation. Experimental evidence is produced which casts doubt on the previously postulated mechanism of reaction, implying the rate limitation of reaction (i) under conditions where chromatographically pure maltose is used as substrate for the enzyme. 2. Experimental

Some effects of local anaesthetics on cell metabolism

Abstract Some effects of local anaesthetics (cocaine, procaine, amethocaine, and cinchocaine) have been investigated. In this study, the local anaesthetics inhibited citrate synthesis in intact cells of Escherichia coli, brain slices, and homogenates. The drugs also inhibited significantly the oxidation of α-oxoglutarate while having no appreciable effect upon the other isolated steps of the tricarboxylic acid cycle. The local anaesthetics also inhibited malate synthesis in intact cells of E. coli, acetoacetate synthesis, and sulfanilamide acetylation in tissue homogenates. In rats subjected to chronic cocaine treatment, there was no effect upon citrate synthesis in vivo nor were there any changes seen in serum and urine lactate levels and serum Ca2+ levels. The drugs had no effect upon any of the metabolic reactions in cellular subfractions or on mitochondrial respiration. The drugs had no effect upon Mg2+stimulated ATPase from rat nerve or on the Na+-sensitive ATPase from rat brain. The results support the probable implication of membrane structure as the site of action of local anaesthetics.

Dual function and common identity of proteins in glycogen metabolism: A hypothesis

Hers, De Wulf and Stalmans have recently reviewed the control of liver glycogen metabolism in the pages of this journal [l] and have offered a scheme for the regulation of the activities of various of the enzymes involved. We have elsewhere proposed an alternative regulatory scheme [2] and present some features of it here as a comment on the hypothesis of Hers et al. As our understanding of the control of glycogen metabolism, first in muscle, and, more recently, in liver has increased, so has the apparent complexity of the process. We feel that the point has been reached where possible simplifications are emerging which remove at least some of the problems that have been posed. There are ten key enzymes involved in the metabolism of the chain-forming 1 + 4-bonds and the regulation of this metabolism, namely: phosphorylase, phosphorylase b kinase, phosphorylase b kinase-kinase, phosphorylase b kinase-phosphatase, phosphorylase a phosphatase, synthetase, synthetase a kinase, synthetase b phosphatase, synthetase b phosphatase-kinase and synthetase b phosphatasephosphatase. In addition, five of these enzymes are reported to exist in two forms. This, in turn, implies the existence of further regulatory enzymes and suggests linear regulatory systems extending indefinitely in either direction. Indeed, the activation of phosphorylase (b + a) by the production of active phosphorylase b kinase, catalyzed in turn by phosphorylase b kinase-kinase, has been referred to as a cascade pro-

The inhibition of choline acetylase by nicotine.

1 Nicotine inhibits the isolated choline acetylase system of brain but has no action on the sulphanilamide acetylating system of liver. 2 Various mechanisms whereby nicotine inhibits acetylcholine synthesis in nerve are discussed.

The regulatory role of amylo-1,6-glucosidase/oligo-1,4-->1,4-glucantransferase in liver glycogen metabolism.

The regulation of mammalian glycogen metabolism in viva has hitherto been evaluated in terms of the well-established regulatory properties of glycogen phosphorylase (EC 2.4.1. l), UDPGglycogen a4glucosyltransferase (EC 2.4.1 .I 1, glycogen synthetase) and associated enzymes [l-3]. This approach cannot be regarded as totally comprehensive in that it fails to take into account the involvement in glycogen metabolism of branching and debranching enzymes specific for cu-l,6-glucosidic linkages. The present investigation was directed towards evaluating the role of the debranching enzyme system, amylo-1,6glucosidase/ oligo-1,4+1,4-glucantransferase (EC 3.2.1.33; EC 2.4.1.25) in the regulation of glycogen metabolism in mammalian liver. Evidence is presented to suggest that this system may assume a rate-limiting function when phosphorylase is subjected to hormonal control by adrenaline.