Calmodulin-dependent protein kinase (kinase II) which is involved in the activation of tryptophan 5-monooxygenase catalyzes phosphorylation of tubulin

Tubulin was shown to be an endogenous substrate of the calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase [T. Yamauchi and H. Fujisawa (1983) Eur. J. Biochem.132, 15–21]. Serine and threonine were identified as the phosphate acceptor amino acids of tubulin. The V_max of the phosphorylation of tubulin and the apparent K_m value for tubulin of calmodulin-dependent protein kinase II were 89 nmol phosphate transferred min^?1 mg kinase II^?1 and 1.7 μm, respectively. The maximum ³²P incorporation into tubulin was 0.18 mol P_i/mol α-tubulin and 0.13 mol P_i/mol β-tubulin. The phosphorylation of tubulin was decreased by the denaturation of tubulin. The phosphorylation of tubulin by kinase II did not affect the assembly of microtubules.     

Estimation of pyruvate,phosphate dikinase activity in maize leaf tissue by (phosphoenolpyruvate plus pyrophosphate)-dependent phosphorylation of AMP

Crude extracts of maize leaf tissue catalysed the phosphorylation of AMP by ³²PPi in the presence of phosphoenolpyruvate (PEP). The reaction was enhanced by F^? and NH₄⁺. The optimum concentrations of AMP, PEP and PPi were 0.3, 10 and 1 mM, respectively. Under these conditions, ca75% of the AMP phosphorylated by ³²PPi was present as ATP and ca25 % as ADP. The activity was reversibly cold labile. The specific activity of crude extracts in the presence of F^? was proportional to enzyme concentration only at protein concentrations < 25,μg/ml. Partially purified pyruvate, phosphate dikinase (PPD) from maize leaf quantitatively phosphorylated AMP to ATP in a (PEP plus PPi)-dependent reaction with the concomitant production of 0.9 mol of pyruvate per mol of AMP phosphorylated. It was concluded that (PEP plus PPi)-dependent phosphorylation of AMP provides a reliable method for estimating PPD activity in crude extracts of maize. Crude maize extracts also catalysed ³²Pi-ATP and ³²PPi-ATP exchange but these activities were not specific for PPD.     

In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ-³²P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation (~25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.     

Inability of the estrogen-induced uterine protein to serve as a substrate for the endogenous phosphokinase(s)

We have studied the phosphorylation of soluble proteins from uterine extracts by an endogenous protein kinase. The analysis of phosphorylation patterns by polyacrylamide gel electrophoresis did not reveal any significant difference in this respect between the soluble proteins from control of 17-β-estradiol stimulated uteri. In both cases, three main components with mol. wt of about 120 000, 60 000 and 45 000 appear preferentially phosphorylated.Estrogen-induced protein did not coincide with any phosphorylated component, although some migrated very closely to it. This was observed whether phosphorylation was performed on uterine extract incubated with [γ-³²P]ATP or on intact organs incubated in the presence of ³²P_i.We conclude that whatever the role of estrogen-induced protein, it is unlikely to be subjected to regulation through the phosphorylation process.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Phosphorylation of neuronal microtubule proteins

Summary Phosphorylation of microtubule protein was tested during differentiation in neuroblastoma cells. Two microtubule proteins were modified, -tubulin and MAP-1 B. In the first case less than one mol of phosphate was incorporated per mol of protein, whereas several residues were phosphorylated in MAP-1 B. The localization of the phosphorylated residue of -tubulin indicated that it is present in an isoform, at its carboxy-terminal region, and probably correspond to the serine 444. When comparing thein vivo phosphorylation of tubulin with that produced by casein kinase IIin vitro, a similar pattern was obtained. A similar result was found upon the comparison of the phosphorylation pattern of MAP-1 B after phosphorylationin vivo andin vitro using casein kinase II. These results suggest a role for casein kinase II in the phosphorylation of microtubule proteins in neuroblastoma cells. A result similar to that found for neuroblastoma cells was found after injection of [³²P]phosphate into the brain of seven-day-old rats; however, a more complex pattern was found for the phosphorylationin vivo in adult rats.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices.

The effect of glucagon on the phosphorylation of pyruvate kinase in ³²P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10^?7 M glucagon, the incorporation of [³²P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10^?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Guanosine triphosphate - dependent autocatalytic phosphorylation of peptide initiation factor 2 from rat liver

If incubated with [γ-³²P]guanosine triphosphate, homogenous peptide initiation factor 2 from rat liver liberates the terminal phosphate from GTP and utilizes 25–40% of it for its own phosphorylation. This phosphorylation is apparently autocatalytic since it does not require any additional proteins. Only the γ-subunit of the factor becomes phosphorylated by an acid-labile bond and the reaction is significantly inhibited by initiator Met-tRNA_F.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.     

Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE

Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser⁸ and Thr¹³⁸ as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser⁹¹, which was phosphorylated by Ca²⁺-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser⁸, Ser⁹¹, and Thr¹³⁸ phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser⁸ was phosphorylated in all p35 molecules, whereas Ser⁹¹ was phosphorylated in 60% and Thr¹³⁸ was phosphorylated in ∼12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.Phosphorylation is a major post-translational modification of proteins, modulating a variety of cellular functions (1, 2). Because most phosphorylation occurs in a highly site-specific manner, identification of phosphorylation sites has been a subject of intense investigation. Several analytical methods have been utilized to identify phosphorylation sites, including mass spectrometry, amino acid sequencing, and radioisotope phosphate labeling of proteins with mutation(s) at putative phosphorylation site(s) (3, 4). Phosphorylation site-specific antibodies are frequently used to detect phosphorylation at target sites (5, 6). Many phosphospecific antibodies are now commercially available. These phosphospecific antibodies are convenient and useful tools for examining site-specific phosphorylation both in vivo and in vitro. However, they are not appropriate for estimating quantitative ratios of phosphorylation states. Electrophoretic mobility shift on SDS-PAGE is also often used to observe phosphorylation (7–10), but this method is not always applied to site-specific phosphorylation.Phos-tag is a newly developed dinuclear metal complex that can be used to provide phosphate-binding sites when conjugated to analytical materials such as acrylamide and biotin (11). In SDS-PAGE using Phos-tag acrylamide, phosphorylated proteins are trapped by the Phos-tag sites, delaying their migration and thus separating them from unphosphorylated proteins. Subsequent immunoblot analysis with phosphorylation-independent antibodies reveals both the phosphorylated and unphosphorylated bands. Because the migration of the phosphorylated proteins is greatly delayed compared with migration in Laemmli SDS-PAGE, it is easy to identify the phosphorylated proteins from observed positions on blots. In the past 3 years, this method has been used to detect phosphorylation states for many proteins such as ERK1/2, cdc37, myosin light chain, eIF2α, protein kinase D, β-casein, SIRT7, and dysbindin-1 (12–21).Cyclin-dependent kinase 5 (Cdk5)¹ is a proline-directed serine/threonine kinase that is expressed predominantly in postmitotic neurons and regulates various neuronal events such as neuronal migration, spine formation, synaptic activity, and cell death (22–24). Cdk5 is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 (25). In addition, Cdk5 activity is deregulated by cleavage of p35 to p25 with calpain, resulting in abnormal activation and ultimately causing neuronal cell death (26–29). Proteolysis of p35, either by proteasomal degradation or cleavage by calpain, is regulated by phosphorylation of p35 by Cdk5 (30–33). Therefore, phosphorylation of p35 is essential for proper regulation of Cdk5 activity and function. We previously identified Ser⁸ and Thr¹³⁸ as major p35 phosphorylation sites (33). We also showed that phosphorylation of p35 decreased during brain development and proposed its relationship to age-dependent vulnerability of neurons to stress stimuli (32). Thus, to understand the in vivo regulation of Cdk5 activity, it is critical to analyze the phosphorylation states of p35 in brain. However, there is no convenient method to analyze the precise in vivo phosphorylation status of the endogenous proteins.In this study, we applied the Phos-tag SDS-PAGE method to analyze the phosphorylation states of p35 in vivo and in cultured neurons. We constructed standard band profiles of phosphorylated p35 by Phos-tag SDS-PAGE using Ala mutants at Ser⁸ and/or Thr¹³⁸. From these experiments, we observed an unidentified in vivo phosphorylation site at Ser⁹¹. We quantified the phosphorylation at each site in cultured neurons and brain, providing the first quantitative estimate of the in vivo phosphorylation states of p35. We discuss the usefulness of Phos-tag SDS-PAGE to analyze the in vivo phosphorylation states of proteins.     

P0 phosphorylation in nerves from normal and diabetic rats: Role of protein kinase C and turnover of phosphate groups

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P₀ phosphate groups was studied in normal and exprimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [³²P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P₀, was increased 2–5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P₀ were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P₀ and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C -isoform. The adenylate cyclase activator, forskolin, had no affect on the PDB-stimulated phosphorylation of P₀ in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P₀ phosphate groups, nerves were incubated with³²P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P₀ from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P₀ phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P₀ phosphate moieties was accelerated in normal, but not in diabetic nerve. These findings clearly establish a prominent role for protein kinase C in P₀ phosphorylation, provide evidence for heterogeneous turnover of P₀ phosphate groups and suggest that cyclic AMP-mediated processes may modulate P₀ phosphorylation. Further, these results indicate that the metabolism of P₀ phosphate moieties is perturbed in nerve from diabetic animals.Special issue dedicated to Dr. Marjoris B. Lees.     

Phosphorylation ofEscherichia coli NADP+-specific glutamate dehydrogenase

Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of³²P-phosphate from [-³²P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.     

On substrate specificity of the donor site of the Escherichia coli ribosomal peptidyl transferase center: Synthesis of dipeptides from 3′-terminal fragments of aminoacyl-tRNA

The insulin receptor of human placenta even after extensive purification is phosphorylated in the presence of [γ-³²P]ATP and NaF, and is dephosphorylated again on incubation in NaF-free medium. Insulin stimulates phosphate incorporation into the M_r95 000 subunit probably by activation of the phosphorylation step. Our data suggest that the insulin receptor contains both kinase and phosphatase activities that may control the phosphorylation state of the receptor.     

Purification and characterization of bovine brain calmodulin-dependent protein kinase II. The significance of autophosphorylation in the regulation of 63 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca²⁺ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca²⁺. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca²⁺ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca²⁺ flux via the protein kinase autophosphorylation mechanism.Abbreviations SDS sodium dodecyl sulfate - EGTA ethylene glycol bis (-aminoethyl ether) - N,N,N,N tetra acetic acid - EDTA ethylenediamine-tetraacetic acid - cAMP cyclic adenosine 35 monophosphate This work is supported by grants from the Medical Research Council of Canada (JHW), the Heart and Stroke Foundation of Alberta (JHW and RKS) and the Heart and Stroke Foundation of Saskatchewan (RKS)     

Isoelectric focusing of phosphorylated cattle rhodopsin

³²P-rhodopsin was partially separated by isoelectric focusing into several fractions of different phosphorylation extent. It was found that the incorporated phosphate is not uniformly distributed in a population of rhodopsin molecules. In a preparation with an average phosphorylation extent of 2.4 moles of phosphate per mole of rhodopsin, most of the ³²P-phosphate was found in fractions where 4–5 phosphates are bound per rhodopsin, whereas a large fraction of the total rhodopsin was not phosphorylated at all. The maximum number of phosphate binding sites in rhodopsin appears to be at least five.Abbreviations used P/Rh moles of phosphate per mole of rhodopsin - ROS rod outer segments Presented in part at the EMBO workshop on Transduction Mechanism of Photoreceptors, held in Jülich, Germany, on 4–8 October, 1976