When a partially purified dog liver phosphorylase phosphatase preparation was incubated with ATP and Mg ions, its activity increased several fold. A slower activation was observed in the presence of Mg ions only. When the same preparation was preincubated with ATP alone, complete inactivation was obtained. The ATP pretreated enzyme could then be reactivated by Mg++, as well as by ATP and Mg++. The activation and the inactivation of the phosphorylase phosphatase, were shown to be dependent on time, temperature and the concentration of ATP and Mg ions. Neither on the activation ncr on the inactivation of the phosphorylase phosphatase could an effect of cyclic 3':5'-AMP be observedThe activity of phosphorylase in liver is of prime importance in glycogen metabolism, and changes rapidly in response to a number of hormonal and other stimuli. The level of phosphorylase activity in liver is to a major extent the result of a balance between the conversion of dephosphophosphorylase to phosphorylase by the enzyme phosphorylase kinase and the reconversion of phosphorylase to dephosphophosphorylase by the phosphorylase phosphatase. Activity changes of the former enzyme have been described in liver [l], and in skeletal muscle [ 2 , 3 ] .In a description of the phosphorylase phosphatase system in bovine adrenal cortex by Merlevede and Riley (41, it has been assumed that this enzyme can exist as an inactive form, which can be converted to the active form by a reaction involving ATP and Mg ions, while inactivation of the active form can be obtained by incubation with ATP alone. A form of the enzyme which appears to be partially active can be obtained by incubation of the enzyme with cyclic 3':5'-AMP in the presence of ATP and Mg ions. Similar observations were made more recently by Chelala and Torres [5] with skeletal muscle phosphorylase phosphatase.During previous studies of the phosphorylase and its regulating enzymes during embryonic development of chicken liver, it was observed [6,7] that all these enzymes could exist in the active or inactive form. Phosphorylase phosphatase from dog liver has been isolated and partially purified (81. However, no activity changes of this enzyme have been described. Since no detailed studies of the activity changes of liver phosphorylase phosphatase are available we have investigated further the activation and inactivation of dog liver phosphorylase phosphatase in an attempt to clarify its role in the regulation of the phosphorylase activity in liver. EXPERIMENTAL PROCEDURE ChemicalsAdenosine triphosphate, adenosine-5'-diphosphate, adenosine-5'-monophosphate and adenosine-3' : 5'-monophosphate (cyclic 3' : 5'-AMP) were obtained from Sigma Chemical Co. (U.S.A.). Stock solutions were prepared by dissolving compounds at neutral pH. EnzymesActive Phosphorylase. The active dog liver phosphorylase used as a substrate for the determinations of phosphorylase phosphatase activity was the second alcohol fraction described by Sutherland (91, and had been dialyzed for about 8 hours against glass distil...

A collection of 655 thermosensitive mutants of Bacillus subtilis 168, obtained by indirect selection, was screened for those lysing at the non-permissive temperature. Thirty-three mutations thus identified were distributed by transformation into eight linkage groups designated lssA to lssH. The distribution was non-random. With the exception of group A, all groups were small, suggesting that mutations identified in each of them may map in one gene only. Linkage groups identified here were mapped in four different regions of the B. subtilis chromosome and their positions relative to reference markers were the following: (i) aroI-lssA-dal-purB; (ii) metC-lssB-lssC-furA-pyrE-cysC-lssD; (iii) lssF-gtaA-lssG-hisA-lssH-cysB; and (iv) cysA-lssE-dnaC-purA. Kinetics of N-acetyl-D-[1-14C]glucosamine incorporation revealed that groups A, B, C, D and F are deficient in peptidoglycan synthesis at the restrictive temperature. In group G, anomalies at the cell wall level were suggested by incorporation and growth curves. It appears that in almost all known cases, thermosensitive lysis mutations in B. subtilis either affect genes involved in peptidoglycan synthesis or lead, more or less directly, to induction of prophages.

Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the rneso-diaminopimelate ( L D -A~~~) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and IssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A,pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group IssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyI-~~-A,pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase I. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapC. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase I, not in the carboxylase, and to belong to the dapC locus. dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A,pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapC genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.

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