Richard H. Haschke

Abstract A fraction containing a protein-glycogen complex was obtained from rabbit muscle by different procedures involving acid precipitation followed by differential centrifugation, direct differential centrifugation, and acetone fractionation. All three preparations were essentially identical in terms of their physical, chemical, and enzymatic characteristics strongly suggesting that the protein-glycogen complex represents a structural and functional unit of the cell rather than artifacts resulting from a given isolation procedure. Electron micrographs of the isolated fractions showed the presence of glycogen granules together with vesicles arising from fragments of the sarcoplasmic reticulum. Sedimentation velocity patterns obtained in the analytical ultracentrifuge, sucrose gradient centrifugation, and Sepharose 2B gel filtration indicated that the material can be separated into a light (approximately 120 S) and a heavy (approximately 600 S) fraction. The light fraction consists mainly of glycogen particles to which are associated phosphorylase, phosphorylase kinase, and phosphatase among other enzymes. The heavy fraction contains mainly the elements of the sarcoplasmic reticulum characterized by a strong ATPase activity and some lysosomes as indicated by the presence of traces of acid phosphatase and amylase activity. Contamination of the glycogen particles by this latter enzyme resulted in their slow degradation that prevented further purification.

Abstract The regulation of enzymatic activity of several enzymes involved in glycogen breakdown has been investigated in a skeletal muscle fraction containing a protein-glycogen complex and elements of the sarcoplasmic reticulum. In this fraction, phosphorylase is entirely in its inactive b form since phosphorylase kinase itself is totally inactive while phosphorylase phosphatase is fully active. Addition of Mg-ATP and Ca2+ triggers an immediate of phosphorylase (b to a conversion) resulting from kinase activation; no occurs with Mg-ATP alone. As soon as all the ATP has been consumed, phosphorylase a is rapidly reconverted to the b form by the phosphatase, and the over-all process can be repeated many times by successive readditions of ATP; this reaction cycle is referred to as flash activation of phosphorylase. The Ca2+ of kinase is reversed by chelation of the metal ion and, therefore, not the result of proteolytic attack by the calcium-dependent kinase-activating factor. Half-maximum of kinase in this system requires 2 x 10-6 m free Ca2+ (in contrast to approximately 10-7 m calcium for purified kinase solutions), i.e. the same Ca2+ concentration needed to trigger muscle contraction. Activation by Ca2+ resulted in a 13-fold increase in affinity of phosphorylase kinase for phosphorylase b. No evidence was obtained that it was mediated or accompanied by a phosphorylation of phosphorylase kinase. Only slight (20%) and delayed of endogenous phosphorylase b was produced by Mg-ATP and 10-5 m cyclic adenosine 3',5'-monophosphate in the absence of added Ca2+, although a 6-fold increase in kinase activity was measured in the usual assay system (at high dilution of phosphorylase kinase and in the presence of purified phosphorylase b). Disruption of the protein-glycogen complex by α-amylase digestion increased the affinity of phosphorylase kinase for Ca2+ 10-fold to the same level observed with the purified enzyme. Readdition of glycogen reversed this effect. Addition of 1 mm glucose-6-P suppressed the flash activation of phosphorylase both by a direct inhibition of the phosphorylase kinase reaction and, indirectly, by increasing the utilization of ATP. Furthermore, activated phosphorylase produced during the reaction was itself partially inhibited by glucose-6-P indicating that phospho-dephospho hybrids (rather than fully phosphorylated phosphorylase a) had been produced. Such hybrids were generated only in the intact (not in the disrupted) protein-glycogen complex.

The authors developed a new method of intrathecal local anesthetic injection in rabbits in order to study the relationship between anesthetic concentration and impaired neurologic function. They found that none of the local anesthetics studied produced persistent neurologic damage in concentrations used clinically. However, lidocaine and tetracaine can be prepared in high concentrations (far exceeding those clinically used) that will produce extensive irreversible neurologic injury and histologic changes. This was also true for sodium bisulfite, an antioxidant used in a number of commercially prepared local anesthetic solutions. Pure solutions of relatively insoluble local anesthetics (bupivacaine and 2-chloroprocaine) failed to produce comparable neurologic or neuropathologic changes when tested at concentrations up to their solubility limits. Extensive neurologic impairment was not necessarily accompanied by equally extensive lesions in the spinal cord and nerve roots.