Detection of ubiquityl-calmodulin conjugates with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues.

The selective degradation of many proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved protein [1]. Ubiquitylated proteins were degraded by the 26S proteasome in an ATP-depended manner. The degradation of ubiquitylated proteins were controlled by isopeptidase cleavage. A well characterised system of ubiquitylation and deubiquitylation is the calmodulin system in vitro [2]. Detection of ubiquityl-calmodulin conjugtates in vivo have not been shown so far. In this article we discuss the detection of ubiquitin calmodulin conjugates in vivo by incubation with a novel high-molecular weight ubiquitylprotein-isopeptidase in rabbit tissues. Proteins with a molecular weight of ubiquityl-calmodulin conjugates could be detected in all organs tested. Incubation with ubiquitylprotein-isopeptidase showed clearly a decrease of ubiquitin calmodulin conjugates in vivo with an origination of unbounded ubiquitin. These results suggest that only few ubiquitin calmodulin conjugates exist in rabbit tissues.

Peri-implant reactivity and osteoinductive potential of immobilized rhBMP-2 on titanium carriers.

Recombinant human BMP-2 (rhBMP-2) was immobilized non-covalently and covalently as a monolayer on plasma vapour deposited (PVD) porous commercially pure titanium surfaces in amounts of 5-8 µg cm(-2), providing a ca. 10-fold increase vs. previously reported values. Dissociation of the immobilized [125I]rhBMP-2 from the surface occurred in a two-phase exponential decay: a first rapid phase (ca. 15% of immobilized BMP-2) with a half-life of 1-2 days and a second slow sustained release phase (ca. 85% of immobilized BMP-2) with a half-life of 40-60 days. Dissociation rate constants of sustained release of k(-1)=1.3-1.9 x 10(-7)s(-1) were determined, allowing an estimation of the binding constants (K(A)) for the adsorbed rhBMP-2 monolayer, to be around 10(12) M(-1). The rhBMP-2-coated surfaces showed a high level of biological activity, as demonstrated by in vitro epifluorescence tests for alkaline phosphatase with MC3T3-E1 cells and in vivo experiments. In vivo osteoinductivity of rhBMP-2-coated implants was investigated in a gap-healing model in the trabecular bone of the distal femur condylus of sheep. Healing occurred without inflammation or capsule formation. The calculated concentration of released rhBMP-2 in the 1mm gap ranged from 20 to 98 nM--well above the half-maximal response concentration (K(0.5)) for inducing alkaline phosphatase in MC3T3-E1 cells. After 4, 9 and 12 weeks the bone density (BD) and bone-to-implant contact (BIC) of the explanted implants were assessed histomorphometrically. Implants with immobilized rhBMP-2 displayed a significant (2- to 4-fold) increase in BD and BIC values vs. negative controls after 4-9 weeks. Integration of implants by trabecular bone was achieved after 4 weeks, indicating a mean "gap-filling rate" of ∼250 µm week(-1). Integration of implants by cortical bone was observed after 9 weeks. Control implants without rhBMP-2 were not osseointegrated. This study demonstrates the feasibility of enhancing peri-implant osseointegration and gap bridging by immobilized rhBMP-2 on implant surfaces which may serve as a model for future clinical applications.

Influence of bone morphogenetic protein-2 on spiral ganglion neurite growth in vitro.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a growth factor of the transforming growth factor-beta superfamily. Members of this protein family are involved in the development of various mammalian tissues, including the inner ear. As their notations indicate, they also have well-known effects on bone formation and regeneration. In this study, we examined the influence of rhBMP-2 on spiral ganglion (SG) neurite growth in vitro and showed the presence of its most preferred receptor BMPR-IB in spiral ganglion cells both in vitro and in vivo. SG explants of postnatal day 4 rats were analysed for neurite length and number after organotypical cell culture for 72 h, fixation and immunolabeling. Different concentrations of rhBMP-2 were used in a serum-free culture media. Neurite growth was compared with control groups that lacked stimulative effects; with neutrophin-3 (NT-3), which is a well-established positive stimulus on neurite length and number; and with combinations of these parameters. The results display that neurite number and total neurite length per explant in particular concentrations of rhBMP-2 increased by a maximum factor of two, while the mean neurite length was not affected. NT-3 demonstrated a much more potent effect, delivering a maximum increase of a factor of five. Furthermore, a combination of both growth factors shows a predominant effect on NT-3. Immunohistological detection of BMPR-IB was successful both in cell culture explants and in paraffin-embedded sections of animals of different ages. The results show that rhBMP-2 is, among other growth factors, a positive stimulus for SG neurite growth in vitro. Most growth factors are unstable and cannot be attached to surfaces without loss of their biological function. In contrast, rhBMP-2 can be attached to metal surfaces without loss of activity. Our findings suggest in vivo studies and a future clinical application of rhBMP-2 in cochlear implant technology to improve the tissue/electrode interface.

A novel nanolayer biosensor principle.

A method for eliminating the mass transport limitation on biosensor surfaces is introduced. The measurement of macromolecular binding kinetics on plane surfaces is the key objective of many evanescent wave (e.g. total internal reflection fluorescence (TIRF)), and surface plasmon resonance (SPR) based biosensor systems, allowing the determination of binding constants within minutes or hours. However, these methods are limited in not being rigorously applicable to large macromolecules like proteins or DNA, since the on-rates are transport limited due to a Nernst diffusion layer of 5-10 microm thickness. Thus, for the binding of fibrinogen (340 kDa) to a surface current SPR biosensors will show a mass transport coefficient of ca. 2 x 10(-6) m/s. In a novel approach with an immiscible fluid vesicle (e.g. air bubble), it has been possible to generate nanoscopic fluid films of ca. 200 nm thickness on the sensor surface of an interfacial TIRF rheometer system. The thickness of the liquid film can be can be easily probed and measured by evanescent wave technology. This nanofilm technique increases the mass transport coefficient for fibrinogen to ca. 1 x 10(-4) m/s eliminating the mass transport limitation, making the binding rates reaction-rate limited. From the resulting exponential kinetic functions, lasting only 20-30s, the kinetic constants for the binding reaction can easily be extracted and the binding constants calculated. As a possible mechanism for the air bubble effect it is suggested that the aqueous fluid flow in the rheometer cell is separated by the air bubble below the level of the Nernst boundary layer into two independent laminar fluid flows of differing velocity: (i) a slow to stationary nanostream ca. 200 nm thick strongly adhering to the surface; and (ii) the bulk fluid streaming over it at a much higher rate in the wake of the air bubble. Surprising properties of the nanofluidic film are: (i) its long persistence for at least 30-60s after the air bubble has passed (2.5s); and (ii) the absence of solute depletion. It is suggested that a new liquid-liquid interface (i.e. a "vortex sheet") between the two fluid flows plays a decisive role, lending metastability to the nanofluidic film and replenishing its protein concentration via the vortices-thus upholding exponential binding kinetics. Finally, the system relaxes via turbulent reattachment of the two fluid flows to the original velocity profile. It is concluded that this technique opens a fundamentally novel approach to the construction of macromolecular biosensors.

A reporter-cell assay for the detection of BMP-2 immobilized on porous and nonporous materials.

Human recombinant bone morphogenetic protein-2 (rhBMP-2) immobilized on the surface of metal implants can facilitate osseointegration. Here, we describe a cell reporter assay useful for quantifying small amounts of immobilized rhBMP-2 on various materials. The peptide was dotted and heat-fixed on titanium, 316L stainless steel, nitrocellulose, or glass, and its distribution was monitored by in situ biotinylation followed by detection with the avidin-biotin method. Bioactivity of rhBMP-2 was demonstrated by means of a confluent layer of osteoblastic MC3T3-E1 cells that evenly covered rhBMP-2-free and rhBMP-2-loaded surface areas, as shown with epifluorescence microscopy of calcein acetoxymethyl (AM)-loaded cells. Expression of osteocalcin, fibronectin, actin, and vimentin increased where cells were located on rhBMP-2 dotted areas, but the signal:noise ratio was too low to bioassay the peptide. However, local pronounced expression of alkaline phosphatase was used to quantify BMP-2 in the range of 5-80 ng/dot by means of a cytochemical color reaction for alkaline phosphatase and image analysis of resulting dots. The lower detection limit was in the order nitrocellulose > glass > titanium > 316L steel. We conclude that the cell reporter assay is useful to assess biological activity of rhBMP-2 even after immobilization on three-dimensional implant materials.

Cytosolic protein ubiquitylation in normal and endotoxin stimulated human peripheral blood mononuclear cells.

The ubiquitin-proteasome pathway is regarded as playing a crucial role in protein breakdown in inflammation and sepsis as well as in the regulation of inflammatory cell responses. In this pathway, ubiquitylation of target proteins is believed to act as a recognition signal for degradation by the 26S proteasome. As yet neither the ubiquitylation rate of cytosolic proteins, as a result of the total ubiquitin-protein ligase (tUbPL) activity, nor the specific ubiquitylation of calmodulin (ubiquitin-calmodulin ligase, uCaM-synthetase) has been determined in human mononuclear cells. Therefore, we studied cytosolic protein ubiquitylation in normal and in endotoxin (LPS)-stimulated human peripheral blood mononuclear cells (PBMNCs).PBMNCs from healthy volunteers were incubated with 0 or 100 ng/ml LPS for 18 h. Cytosolic extracts were obtained by hypotonic lysis and ultracentrifugation. TUbPL was measured as [(125)I]-[CT]-ubiquitin incorporation into the sum of cytosolic proteins. UCaM-synthetase activity was quantified with the fluphenazine (FP)-Sepharose affinity adsorption test. Endotoxin stimulation appears to inhibit tUbPL 3.7 +/- 2.7-fold to 48 +/- 43 fkat/mg (n = 6). UCaM-synthetase in cultures (n = 5) without endotoxin was determined to be 91 +/- 32 fkat/mg +Ca(2+) and 29 +/- 23 fkat/mg -Ca(2+). With endotoxin uCaM-synthetase was 138 +/- 73 fkat/mg +Ca(2+) and 14 +/- 22 fkat/mg -Ca(2+). Ca(2+)-specificity (ratio +/- Ca(2+)) of uCaM-synthetase increases from 3.1 without LPS to 10 after LPS stimulation, which was caused by a 2-fold decrease in minus Ca(2+) activity and a 1.5-fold increase in plus Ca(2+) activity. The data indicate specific regulatory effects of endotoxin on the cytosolic ubiquitylation systems in human PBMNCs.

Modulation of calmodulin function by ubiquitin-calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin.

Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell-free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl-calmodulin synthetase (ubiquitin-calmodulin ligase, EC 6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+-binding loop of calmodulin and has the octapeptide structure -L-F-D-K21-D-G-D-G- with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl-calmodulin synthetase. Removal of the 41 C-terminal amino acids (fourth Ca2+-binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site-specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site -V-F-D-K94-D-G-N-G- in the third Ca2+-binding loop is apparently not ubiquitylated by the synthetase.

The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the calmodulin-binding second component and enzymatic properties.

Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.

The ubiquityl-calmodulin synthetase system from rabbit reticulocytes: isolation of the ubiquitin-binding first component, a ubiquitin-activating enzyme.

Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.

Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues.

Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40,000 x g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4-80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3-4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse of 30 min was followed in liver crude extracts by the addition of EGTA, which specifically inhibits ubiquityl-calmodulin synthesis, a half-life of calmodulin-conjugate decay of 15-20 min is observed. A similar conjugate half-life of ca. 30 min was observed after addition of EDTA excluding that conjugate decay is due to an ATP-dependent proteolytic process. Studying the decay of purified ubiquitin-125I-BH-calmodulin conjugates in cell-free reticulocyte extracts led to the discovery of an ATP-independent isopeptidase activity which splits ubiquitin-calmodulin conjugates without leading to detectable calmodulin fragments. The rapid decay of ubiquitin-calmodulin conjugates in tissue extracts can therefore be plausibly explained by a ubiquityl-calmodulin splitting isopeptidase activity.

Monitoring fibrinogen adsorption kinetics by interfacial TIRF rheometry.

The adsorption kinetics of purified fibrinogen to unmodified and aminopropylsilane-modified quartz glass surfaces were studied under pseudo-first order (binding-unit excess) conditions by the total internal reflection fluorescence (TIRF) method. Fluorescence in the adsorbed protein layer (350 nm) was excited by the evanescent wave at 285-290 nm. In order to reduce and possibly eliminate the influence of mass transfer on the kinetics of fibrinogen adsorption, a novel protein adsorption chamber containing a cone-and-plate rheometer with total internal reflection technology was employed. The aim of the study was to obtain critical shear rates, at which the adsorption rate of fibrinogen became independent of diffusion. Therefore, shear rates were varied between 0 and 7200 s-1 at initial fibrinogen concentrations of c9 = 4.7 and 17.7 micrograms mL-1. The adsorption rate of fibrinogen increased 5-17-fold, depending on the surface, as the critical shear rate was approached. Above the critical shear rates the kinetic data of fibrinogen adsorption could be fitted at c9 = 4.7 micrograms mL-1 to a single exponential function, indicating the predominance of a single binding step with a half-life of ca 20 s. At the higher initial concentration of c9 = 17.7 micrograms/mL-, however, a significant deviation from the single exponential behavior was observed in the first 10 s of the adsorption reaction, indicating a very fast initial event with a half-life of ca 5 s in addition to a slower binding reaction with a half-life of ca 35 s. Thus the novel TIRF rheometer can resolve kinetics down to half-lives of 5 s and possibly even lower.

Analysis of affinity supports by 13C CP/MAS NMR spectroscopy: application to carbonyldiimidazole- and novel tresyl chloride-synthesized agarose and silica gels.

A major problem in affinity technology is the analysis of the synthesized solid supports, since liquid phase methodology can generally not be applied. Recently we reported a novel reaction sequence for the 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) coupling of nucleophiles [Demiroglou et al. (1994) Angew. Chem. Int. Ed. Engl. 33, 120-123] to agarose and concluded that previously proposed structures could not be correct. However it was not possible for us to conclusively define the new reaction products because the agarose derivatives could not be solubilized for customary liquid state 13C NMR analysis. Therefore in this paper solid state 13C CP/MAS NMR spectroscopy is applied for the first time to the polysacharide agarose. Using alkyl agarose derivatives prepared by the carbonyldiimidazol method as control we found that reliable spectra in agreement with the published reaction mechanism could be obtained. The method was then applied to the novel reaction products of the tresyl chloride reaction. From the solid state 13C NMR spectra and other quantitative data it is concluded that a beta-sulfonyl carboxylic acid is generated during alkaline hydrolysis of tresyl agarose and that alkyl amines are coupled by a beta-sulfonyl amide bond in an elimination-addition reaction in the absence of SO- and SC-scission of the tresyl group. In the case of alkane thiol coupling the absence of SC-scission cannot be demonstrated indicating either a different reaction mechanism or possibly a mixture of reaction products.

Ubiquitin and the enigma of intracellular protein degradation.

Contrary to widespread belief, the regulation and mechanism of degradation for the mass of intracellular proteins (i.e. differential, selective protein turnover) in vertebrate tissues is still a major biological enigma. There is no evidence for the conclusion that ubiquitin plays any role in these processes. The primary function of the ubiquitin-dependent protein degradation pathway appears to lie in the removal of abnormal, misfolded, denatured or foreign proteins in some eukaryotic cells. ATP/ubiquitin-dependent proteolysis probably also plays a role in the degradation of some so-called 'short-lived' proteins. Evidence obtained from the covalent modification of such natural substrates as calmodulin, histones (H2A, H2B) and some cell membrane receptors with ubiquitin indicates that the reversible interconversion of proteins with ubiquitin followed by concomitant functional changes may be of prime importance.

Cyanogen bromide and tresyl chloride chemistry revisited: the special reactivity of agarose as a chromatographic and biomaterial support for immobilizing novel chemical groups.

The cyanogen bromide and tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) methods belong to the best-known activation procedures for solid supports in biochemistry. In both cases the originally proposed reaction mechanisms were revised many years later. In this paper important aspects of these two major activation reactions in connection with the singular polysaccharide support, agarose, will be treated with emphasis on the novel reaction mechanism recently proposed for tresyl chloride. In addition, the special role played by sulfur in the new uncharged alkyl-S-S-gels is examined in connection with the phenomenon of base-atom recognition.

Interaction sites on phosphorylase kinase for calmodulin.

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the alpha subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the beta subunit and a peptide from the alpha subunit present in a region deleted in the alpha' isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca2+ binding to calmodulin in presence of peptides was measured. By this way, the peptides alpha 542-566, alpha 547-571, alpha 660-677 and beta 597-614 have been found to bind specifically to calmodulin. Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the alpha and beta subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in gamma as well as to two regions in alpha and beta. Exogenous calmodulin can bind to two regions in alpha and in beta. A binding stoichiometry of 0.8 mol of calmodulin/alpha beta gamma delta promoter of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23 nM calmodulin which is in the affinity range of calmodulin binding peptides from beta to calmodulin. Therefore, binding of exogenous calmodulin to beta activates the enzyme. A model for switching endogenous calmodulin between alpha, beta and gamma and modulation of ATP binding to alpha as well as Mg2+/ADP binding to beta by calmodulin is presented.

The binding of phosphorylase kinase to immobilized calmodulin.

The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5-5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca2+ concentrations (5-10 microM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.