Jerry H. Wang

Effect of Ca2+ on the stability of the protein activator of cyclic nucleotide phosphodiesterase

Mammalian cyclic nucleotide phosphodiesterase exists in multiple molecular forms [l] . One of these forms depends on a protein activator for full activity [2-41 . Recently, the protein activator of cyclic nucleotide phosphodiesterase has been purified to homogeneity [5-71. Studies from several laboratories have established that the enzyme activation has the following properties: a) the activation of the enzyme by its protein activator requires the presence of Ca2+ [6-91, b) the enzyme and the protein activator associate to form a protein complex in the presence of Ca2+ [lo121 and c) the protein activator binds Ca2+ with a high affinity [6-81. Based on these observations, it has Leen postulated that the free protein exists in an inactive conformation which may be converted to an active state upon binding of Ca2’. Direct evidence in support of a Ca2+ induced conformational change in the protein activator has not been reported. In the present study, it is shown that Ca2+ exerts a significant effect on the stability of the protein activator. Especially, the resistance of the protein activator to tryptic or chymotryptic attack is greatly enhanced by Ca”‘. Concentrations of Ca2+ effective for these protections are in the same range as those required to show Ca2+ binding to the protein activator. The results lend support to the suggested role of Ca2’ in the enzyme activation. A preliminary account has been presented [ 121.

Inhibition of calmodulin-activated cyclic nucleotide phosphodiesterase by triton x-100

Summary The activity of calmodulin-activated cyclic nucleotide phosphodies-terase in a standard reaction containing 40 ng calmodulin could be inhibited about 50% by 14 μM Triton x-100. In contrast, the calmodulin-independent phosphodiesterase required a much higher concentration of the detergent, 380 μM for 50% inhibition of its activity. The potent inhibitory effect of Triton X-100 on the calmodulin-activated phosphodiesterase reaction could be reversed by high concentrations of calmodulin. Using the gel filtration technique, it was demonstrated that 3 H-Triton X-100 bound to calmodulin with high affinity in buffer containing Ca 2+ . The binding of the detergent to calmodulin was mostly eliminated in the presence of trifluoperazine, a neuroleptic drug. Among other detergents examined, including Lubrol WX, Triton QS and Triton CF, only Triton CF exhibited preferential inhibition of the calmodulin-activated phosphodiesterase. The results suggest that certain detergents in the Triton family inhibit calmodulin action by undergoing Ca 2+ -dependent binding to the protein at the neuroleptic drug site.

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

Abstract A cyclic nucleotide- and Ca2+-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. 254 , 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca2+-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.

Dependence on the order of addition of MAPs and GTP for microtubule assembly

proteins, known as microtubule-associated proteins (MAPS) (review [l-3]). Since the assembly is inhibited by low tem- peratures the process is often initiated by warming a cold solution of tubulin, MAPS and GTP to 37°C [4-61. An alternative method of initiating the micro- tubule assembly involves the addition of GTP to a pre-warmed solution containing tubulin and MAPS [7]. Here, we have observed that both the rate and the extent of the microtubule assembly process are dependent on the order of addition of GTP and MAPS to the 2 min pre-warmed tubulin solution. The results suggest that the 2 agents undergo a time- dependent interaction in promoting tubulin polym- erization. 1/5th of the supernatant, and incubated at 4°C for 30 min. After incubation, it was centrifuged at 37 000 rev./min for 60 min in a T50.1 Beckman rotor at 4°C. The 2 subsequent polymerization-depolym- erization steps were carried out in the absence of glycerol. Tubplin was separated from MAPS by buffer A- equilibrated phosphocellulose chromatography as in [9]. Phosphocellulose-eluted MAPS were desalted by gel filtration on buffer A-equilibrated Sephadex G-25 column. Tubulin and MAPS were either used imme- diately or frozen by liquid nitrogen and stored in -70°C freezer for no more than 2 weeks. SDS-gel electrophoretic analysis indicated that tubulin samples were homogeneity and that the MAPS contained mainly high M, proteins with low MI contamination. 2. Materials and methods Polymerization of tubulin was initiated by the 2 methods described. Turbidity was measured as an indication on the amount of polymerization at an absorbance of 350 nm in a 37°C