The substructure of phosphodiesterase as established by radiation inactivation: A reinterpretation of results

A model for the activation of phosphodiesterase by calmoduling based on a conversion of inactive dimers to active monomers, derived from radiation inactivation studies J. Biol. Chem. (1981) 256, 11351–11355 has been re-examined using a simple probability argument. We conclude that the original model is not supported by the radiation inactivation studies, since our analysis of this model would predict that the rate of radiation inactivation of calmodulin-dependent phosphodiesterase activity be exactly twice that for the decay in total activity in marked contrast with the results obtained.     

Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation

The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approximately 60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approximately 105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiations destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity.     

Measurement of protein activator levels in experimental diabetic rat adipose tissue.

Experimental diabetes induced by streptozotocin has been shown to decrease the level of cyclic AMP phosphodiesterase activity in rat adipose tissue. This reduced activity was restored with insulin. Protein activator, a small molecular weight substance, is essential for full activity of some component phosphodiesterases. Herein we demonstrate a significant decrease in protein activator level in the 13,000 X g boiled supernatant from streptozotocin-diabetic rat adipose tissue. However, although a decrease in protein activator level is consistent with diabetic inactivation of phosphodiesterase activity, additional studies presented here suggest that a defect in the diabetic phosphodiesterase enzyme itself also contributed to the decrease of total phosphodiesterase activity.     

Concepts of the oligomeric structure and function of Na, K-ATPase based on the results of studying ligand binding and of determining the size of the molecular target

This review is devoted to the discussion of the sizes and molecular structure of the minimal functional unit of Na, K-ATPase. Special attention is paid to the data obtained by radiation inactivation method and studies on ligand binding. The model for the stepwise radiation inactivation of Na, K-ATPase is proposed. The conclusion is drawn that Na, K-ATPase has a dimeric structure, the interactions between its alpha-subunits stabilize the quaternary structure of the pump. Functionally, each alpha-subunit in a stabilized structure possesses a full hydrolytic activity.     

Involvement of ATP in activation and inactivation sequence of phosphodiesterase in frog rod outer segments

Cyclic GMP phosphodiesterase in frog rod outer segments is activated after flash illumination and is inactivated when left in the dark. ATP reduces the initial peak activity caused by dim flashes (with 50 microM ATP being required for a half-maximal effect) and also accelerates inactivation (with 2 microM ATP being required for a half-maximal effect). An acceleration of inactivation caused by ATP addition is 3-7-fold, depending on the preparation, and ATP effect can be observed even 1 min after a dim flash is given. The accelerated inactivation is also flash intensity-dependent. A low intensity of light causes more rapid inactivation than does a high intensity of light. ATP appears to control phosphodiesterase activity in various ways.     

3',5' Cyclic nucleotide phosphodiesterase from human platelets: effect of heat upon the multiple forms and their interconversion

A 33,000 g supernatant from human platelets showed a biphasic heat inactivation curve at 45, 50 and 55 degrees C of the cAMP and cGMP phosphodiesterase. This could suggest the presence of two differently heat sensitive phosphodiesterases. However, a preparation heated for 30 min at 55 degrees C, where only the apparently thermostable form of the enzyme remained, still displayed the same characteristics as the starting material, i.e. two apparent Km values for cAMP, a cAMP specific activity lower at low protein concentration (less than 50 micrograms/ml) than at high protein concentration(greater than 100 micrograms/ml), and three peaks of activity upon linear sucrose density gradient. Moreover, a biphasic inactivation curve was again observed after a second heat treatment. These results demonstrated that the heat effect is not a simple protein denaturation of one of two independent species. A study at different temperatures of the profile of the cAMP phosphodiesterase upon sucrose gradient demonstrated that the dissociated form was predominant at high temperature whereas lower temperature favored the associated form. During heat treatment, the dissociated form is at first denatured and this leads to a shift in the equilibrium between the associated and dissociated forms of the phosphodiesterase in favor of the dissociated form. From the overall results, one can draw a model for phosphodiesterase regulation by dissociation-reassociation.     

Characterization of the functional domains of the natriuretic peptide receptor/guanylate cyclase by radiation inactivation.

Radiation inactivation has been used to evaluate the molecular size of domains responsible for atrial natriuretic peptide (ANP)-binding and cyclase functions of the ANP receptor/guanylate cyclase. Two types of inactivation curves were observed for cyclase function in both adrenal cortex and aortic smooth muscle cells: 1) biphasic with enhanced guanylate cyclase activity after exposure to low radiation doses and 2) linear after preincubation of membrane proteins with 0.5 microM ANP or solubilization with Triton X-100. The existence of an inhibitory component was the simplest model that best explained the types of radiation curves obtained. Activation of guanylate cyclase by ANP or Triton X-100 could occur via the dissociation of this inhibitory component from the catalytic domain. On the other hand, the loss of ANP-binding activity was linear with increasing radiation exposures under basal, ANP treatment, and Triton X-100 solubilization conditions. Radiation inactivation sizes of about 30 kDa for cyclase function, 20 kDa for ANP-binding function, and 90 kDa for inhibitory function were calculated. These studies suggest that the ANP receptor/guanylate cyclase behaves as a multidomain protein. The results obtained by radiation inactivation of the various biological functions of this receptor are compatible with the hypothesis of an intramolecular inhibitory domain repressing the guanylate cyclase catalytic domain within its membrane environment.     

A monomer-dimer model explains the results of radiation inactivation: binding characteristics of insulin receptor purified from human placenta

The technique of radiation inactivation has been used on highly purified human placental insulin receptor in order to determine the functional molecular size responsible for the insulin binding and to evaluate the "affinity regulator" hypothesis, which has been proposed to explain the increase in specific insulin binding to rat liver membranes observed at low radiation doses [Harmon, J. T., Hedo, J. A., & Kahn, C. R. (1983) J. Biol. Chem. 258, 6875-6881]. Three different types of inactivation curves were observed: (1) biphasic with an enhanced binding activity after exposure to low radiation doses, (2) nonlinear with no change in binding activity after exposure to low radiation doses, and (3) linear with a loss in the binding activity with increasing radiation exposures. A monomer-dimer model was the simplest model that best described the three types of radiation inactivation curves observed. The model predicts that an increase in insulin binding activity would result after exposure to low radiation doses when the initial dimer/monomer ratio is equal to or greater than 1 and a monomer is more active than a dimer. The monomer size of the binding activity was estimated to be 227,000 daltons by this model. This value most likely reflects the size of the monomeric alpha beta form. To substantiate this model, the purified receptor was fractionated by Sepharose CL-6B chromatography. The insulin binding profile of this column indicated two peaks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity

The nucleotide-sugar pyrophosphatase activity of rat small intestine was studied using GDP-[14C]Man as substrate. The highest specific activities in the gastrointestinal tract were in the proximal small intestine, with a preferential localization in villus tip cells. Purified brush-border membranes were highly enriched in nucleotide-sugar pyrophosphatase. After the enzyme was solubilized with detergent and purified 180-fold, it hydrolyzed FAD and p-nitrophenyl-5'-thymidylate, as well as nucleotide sugars. That the same enzyme, a 5'-nucleotide phosphodiesterase, is responsible for nucleotide-sugar pyrophosphatase, phosphodiesterase I, and FAD pyrophosphatase activities is indicated by: co-migration in electrophoresis, parallel thermal inactivation, competitive inhibition studies, and similar regional, cellular, and subcellular localizations.     

Inhibition of Dna Synthesis In the Macronuclear Replication Band of Euplotes Eurystomus

ABSTRACT. The replication band is a large, migrating, macronuclear domain that is the site of DNA synthesis in hypotrichous ciliated protozoa. A number of agents that produce inactivation of this structure and its replicational activity are described here. These agents include heat shock, aphidicolin, cell crowding, various cAMP phosphodiesterase inhibitors and a calmodulin inhibitor. With the exception of aphidicolin, which has a direct inhibitory effect upon DNA polymerases, the mechanisms of inactivation are presently unknown. the inactivating properties of cAMP phosphodiesterase inhibitors suggest that intracellular cAMP levels may influence replication band structure and function.     

Molecular size of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase as determined in situ in Golgi membranes by radiation inactivation.

The radiation inactivation method was used to determine the molecular size of the two enzymes that participate in the synthesis of the phosphomannosyl recognition marker of lysosomal proteins. The determinations were carried out in situ, in Golgi membranes isolated from normal human placenta and cultured skin fibroblasts. A molecular size of 228 +/- 29 kDa was found for placental N-acetylglucosaminyl-phosphotransferase, and 129 +/- 11 kDa for placental alpha-N-acetylglucosaminyl phosphodiesterase. The values for the fibroblast enzymes were about 20% higher, 283 +/- 27 kDa and 156 +/- 14 kDa for the transferase and phosphodiesterase respectively. Triton X-100 had no effect on the molecular size of these enzymes.     

Radiation inactivation experiments predict that a large aggregate form of the insulin receptor is a highly active tyrosine-specific protein kinase

The technique of radiation inactivation has been used on a highly purified insulin receptor in order to determine the functional molecular size responsible for tyrosine-specific protein kinase activity. When both insulin binding and kinase activities were analyzed with the same receptor preparations, the functional size for kinase activity was found to be larger than that for insulin binding activity. The radiation inactivation curve for kinase activity was multiphasic. This indicates that at least two components contribute to total kinase activity. The average minimal functional size for the kinase was 370,000 +/- 60,000 daltons (n = 7) which corresponds to the alpha 2 beta 2 form of the insulin receptor. The average functional size for larger forms was estimated to be approximately 4 X 10(6) daltons. (To minimize the complexity of the model used in this analysis, we have analyzed the radiation inactivation curves of the insulin receptor kinase activity with a two-component model. However, we believe that the larger component, greater than 1 X 10(6) daltons, is probably not a single molecular weight species but rather represents a continuum of sizes or aggregates of the alpha 2 beta 2 form of the receptor.) These larger forms contributed 93% of the total activity. Mild reduction of the insulin receptor preparation with dithiothreitol (DTT) activated the total kinase activity by 3.5-fold. Under this condition, the minimal functional kinase size was 380,000 +/- 30,000 daltons (n = 6) while the average functional size for the larger forms was approximately 3 X 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of 2 forms of cyclic nucleotide phosphodiesterase from the rat brain under normal conditions and after irradiation

It is established that the functional activity of two phosphodiesterase forms--phosphodiesterase I (Ca2+-calmodulin-sensitive) and phosphodiesterase II (Ca2+-calmodulin-insensitive), isolated from grey matter of the irradiated rat brain varies essentially in comparison with that of the normal rats. In the early period of acute radiation injury both phosphodiesterase I sensitivity to calmodulin and phosphodiesterase II special activity under hydrolysis of 3', 5'-GMP decrease but phosphodiesterase I special activity under hydrolysis of 3', 5'-GMP increases. The investigation of temperature dependence of phosphodiesterase I and phosphodiesterase II activations revealed changes in character of curves, the temperature optimum under irradiation being unchanged and inflections appearing on the Arrhenius curves.     

Involvement of arginine residues in the activation of calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase

Pretreatment of an affinity-purified, brain calmodulin (CaM)-dependent phosphodiesterase (EC 3.1.4.17) with p-hydroxyphenylglyoxal (pHPG), a specific arginine-modifying reagent, resulted in a time-dependent loss in CaM-stimulated hydrolysis of cyclic AMP and cyclic GMP with no change in basal, CaM-independent activity. The loss in CaM-stimulated activity was preceded by a transient increase in CaM-dependent activity. Phenylglyoxal was 10-fold more effective than pHPG in promoting the loss of CaM-stimulated activity with a second-order rate constant of 13.3 M-1 min-1. Other arginine-modifying reagents, 1,2-cyclohexanedione and 2,3-butanedione, were not effective. The pHPG-modified enzyme was activated by 100 microM lysophosphatidylcholine to levels comparable to CaM-stimulated activity. The arginyl-modified enzyme was also activated by chymotrypsin and trypsin but not to the extent of the untreated enzyme stimulated with CaM. The presence of CaM during chemical modification with pHPG protected the enzyme from inactivation. Both the extent of activation and the amount of CaM necessary for 50% maximal activation were affected by pHPG treatment of the enzyme. The approximate number of modified arginines estimated by [7-14C]phenylglyoxal incorporation and amino acid analysis after complete inactivation of CaM stimulation was seven residues per catalytic subunit assuming enzyme homogeneity. The Stokes radius and sedimentation coefficient of the enzyme were unchanged by the modification. These results suggest that arginine residues are critical for functional interaction between phosphodiesterase and CaM and that controlled modification can selectively alter CaM-stimulated enzyme activity.     

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.     

Rate of inactivation of adenyl cyclase and phosphodiesterase: determinants of brain cyclic AMP

In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.     

同步辐射软X射线对枯草杆菌的诱变效应

采用同步辐射软X射线对枯草芽孢杆菌(Bacillus subtilis)1831菌株进行辐照处理,研究了不同剂量下3．1nm的软X射线对其芽孢的失活和诱变作用。结果表明：同步辐射软X射线对枯草杆菌芽孢的剂量存活曲线表现为典型的“肩型”,对芽孢的失活作用属于“单靶多击”方式,失活击中数等于3。根据脱脂牛奶平板上蛋白酶活力大小的测量统计,以变异系数作为诱变效应指标,软X射线对芽孢具有一定的诱变作用。