Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.

The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.

Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione.

The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (alphabetagammadelta)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: alphabeta, betagammagamma, alphagamma and a doublet of differently migrating homodimers, betabeta1 and betabeta2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of alphagamma was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of betagammagamma formed, with Mg2+ having the greatest effect, and all except two increased the amount of betabeta1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.

The inhibitory effects of N omega-nitro-L-arginine methyl ester on nitric oxide synthase activity vary among brain regions in vivo but not in vitro.

We studied the effects of intracerebroventricular and intraperitoneal injection and the in vitro effects of N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the nitric oxide synthase activities of the cerebellum, brainstem, hypothalamus, hippocampus, and the remainder of the brain after dissections. Male rats were chronically implanted with lateral icv guide cannula. L-NAME was injected in doses of 0.2, 1, and 5 mg intracerebroventricularly, and 50 mg/kg intraperitoneally. L-NAME induced dose-dependent suppression of NOS activities in each brain region. The threshold dose was 0.2 mg; 1 mg L-NAME completely abolished brain nitric oxide synthase activity 90 min after the injection. Brain NOS activities returned to baseline level 48 h after the injection of 5 mg L-NAME. There were significant differences between the sensitivity of various regions to L-NAME after in vivo but not in vitro administration of the enzyme inhibitor. These findings indicate that intracerebroventricular injection of L-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo. The differences between the sensitivity of different brain regions to L-NAME as well as the relative fast recovery of nitric oxide synthase activities must be taken into account when L-NAME is administered intracerebroventricularly to rats.

Circadian variation of nitric oxide synthase activity and cytosolic protein levels in rat brain.

The circadian variation of nitric oxide synthase (NOS) activity and cytosolic protein content in the cerebellum, brainstem, hypothalamus, hippocampus, and the remainder of the brain were studied in rats. Both NOS activity and cytosolic protein concentrations were the highest during the dark period and lowest in the light period. Hypothalamic NOS activity exhibited the most pronounced change in activity with time increasing by approximately 120% from mid-light to mid-dark.