Native Regulatory Subunit Research Articles

Mammalian Brain Ca2+ Channel Activity Transplanted into Xenopus laevis Oocytes

Several mutations on neuronal voltage-gated Ca2+ channels (VGCC) have been shown to cause neurological disorders and contribute to the initiation of epileptic seizures, migraines, or cerebellar degeneration. Analysis of the functional consequences of these mutations mainly uses heterologously expressed mutated channels or transgenic mice which mimic these pathologies, since direct electrophysiological approaches on brain samples are not easily feasible. We demonstrate that mammalian voltage-gated Ca2+ channels from membrane preparation can be microtransplanted into Xenopus oocytes and can conserve their activity. This method, originally described to study the alteration of GABA receptors in human brain samples, allows the recording of the activity of membrane receptors and channels with their native post-translational processing, membrane environment, and regulatory subunits. The use of hippocampal, cerebellar, or cardiac membrane preparation displayed different efficacy for transplanted Ca2+ channel activity. This technique, now extended to the recording of Ca2+ channel activity, may therefore be useful in order to analyze the calcium signature of membrane preparations from unfixed human brain samples or normal and transgenic mice.

Regulation-defective mutants of type I cAMP-dependent protein kinase. Consequences of replacing arginine 94 and arginine 95

The type I alpha regulatory subunits of cAMP-dependent protein kinase contain an autoinhibitor site, Arg94-Arg-Gly-Ala-Ile, which serves as a pseudosubstrate. To evaluate their contribution to subunit association, Arg94 and Arg95, key determinants for peptide recognition, were replaced singly and in tandem with Ala, Glu, and His. Unlike substrate peptides in which replacement of either arginine leads to an increase in Km of approximately 3 orders of magnitude, replacement of either arginine causes only a maximal 20-fold decrease in subunit association. Replacement of both arginine residues with alanine, however, generates a regulatory subunit that can no longer recombine with the catalytic subunit under physiological conditions when the regulatory subunit is saturated with cAMP. To evaluate more fully the specific consequences of replacing these 2 arginine residues, a rapid gel filtration chromatographic method was developed so that subunit affinity could be measured independently of assaying for catalytic activity. The R94,95A mutant shows a Kd(app) = 677 nM, representing an increase of greater than 3 orders of magnitude compared with the native subunits in the presence of MgATP. In the absence of MgATP, the Kd(app) for native regulatory subunit was 125 nM, whereas the Kd(app) for the R94,R95A mutant regulatory subunit was determined to 2.87 microM. When this mutant holoenzyme is assayed at microM concentrations, no activity is observed, whereas below microM, activity is observed because of cAMP-independent subunit dissociation.

Dissecting the domain structure of the regulatory subunit of cAMP-dependent protein kinase I and elucidating the role of MgATP.

A truncated regulatory subunit of cAMP-dependent protein kinase I was constructed which contained deletions at both the carboxyl terminus and at the amino terminus. The entire carboxyl-terminal cAMP-binding domain was deleted as well as the first 92 residues up to the hinge region. This monomeric truncated protein still forms a complex with the catalytic subunit, and activation of this complex is mediated by cAMP. The affinity of this mutant holoenzyme for cAMP and its activation by cAMP are nearly identical to holoenzyme formed with a regulatory subunit having only the carboxyl-terminal deletion and very similar to native holoenzyme. The off rate for cAMP from both mutant regulatory subunits, however, is monophasic and very fast relative to the biphasic off rate seen for the native regulatory subunit. The effects of NaCl, urea, and pH on cAMP binding are also very similar for the mutant and native holoenzymes. Like the native type I holoenzyme, both mutant holoenzymes bind ATP with a high affinity. The positive cooperativity seen for MgATP binding to the native holoenzyme, however, is abolished in the double deletion mutant. The Hill coefficient for ATP binding to this mutant holoenzyme is 1.0 in contrast to 1.6 for the native holoenzyme. The Kd (cAMP) is increased by approximately 1 order of magnitude for both mutant forms of the holoenzyme in the presence of MgATP. A similar shift is seen for the native holoenzyme. Further characterization of the MgATP-binding properties of the wild-type holoenzyme indicates that a binary complex containing catalytic subunit and MgATP is required, in particular, for reassociation with the cAMP-bound regulatory subunit. This binary complex is required for rapid dissociation of the bound cAMP and is probably responsible for the observed reduction in cAMP-binding affinity for the type I holoenzyme in the presence of MgATP.

Communication between polypeptide chains in aspartate transcarbamoylase. Conformational changes at the active sites of unliganded chains resulting from ligand binding to other chains.

A largely inactive derivative of the catalytic subunit of Escherichia coli aspartate transcarbamoylase containing trinitrophenyl groups on lysine 83 and 84 was used to study communication between polypeptide chains in the holoenzyme and the isolated catalytic trimers. Addition of native regulatory dimers to the derivative yielded a holoenzyme-like complex of low activity which exhibited sigmoidal kinetics and was inhibited by CTP and activated by ATP. The binding of CTP and ATP to the regulatory subunits caused significant and opposite changes in the absorption spectrum resulting from changes in the environment of the sensitive chromophores at the active sites. In allosteric hybrid molecules containing one native and one trinitrophenylated catalytic subunit, along with native regulatory subunits, the binding of a bisubstrate analog, N-(phosphonacetyl)-L-aspartate, to the native catalytic subunit resulted in a perturbation of the spectrum of the chromophore on the unliganded modified chains. Thus the conformational changes associated with the allosteric transition responsible for both heterotropic and homotropic effects are propagated from the sites of ligand binding to the active sites of unliganded distant chains. In addition to the communication from regulatory chains to catalytic chains and the cross-talk from one catalytic subunit to the other, communication between individual catalytic chains in isolated trimers was also demonstrated. By constructing hybrid trimers containing one trinitrophenylated chain and two native chains, we could detect a change in the environment of the chromophore upon the binding of the bisubstrate analog to the native chains.

19F nuclear magnetic resonance studies of communication between catalytic and regulatory subunits in aspartate transcarbamoylase.

19F nuclear magnetic resonance (NMR) spectroscopy was used to study "communication" between the catalytic and regulatory subunits in aspartate transcarbamoylase of Escherichia coli. Hybrid enzymes composed of fluorotyrosine-labeled regulatory subunits and native catalytic subunits or of native regulatory subunits and fluorotyrosine-labeled catalytic subunits were constructed and shown to have the allosteric kinetic properties of native enzyme. These hybrids exhibited the ligand-promoted "global" conformational changes characteristic of native aspartate transcarbamoylase and alterations in the NMR spectrum when ligands bind to the active site. The NMR difference spectrum caused by the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to the hybrid containing 19F-labeled regulatory chains consisted of two troughs and a peak, suggesting that two tyrosines in the regulatory polypeptide chains were affected by the binding of ligand to the catalytic subunits. The increase in magnitude of the peak appeared to depend directly on the fractional saturation of the active sites. A peak with two distinct shoulders was observed in the 19F NMR spectrum of the hybrid containing fluorotyrosine in the catalytic chains when it was saturated with the ligand, whereas the spectrum for the unliganded enzyme consisted of a single peak. The NMR difference spectrum showed that the bisubstrate ligand perturbed at least two resonances, and these changes appeared to be tightly linked to the binding of the ligand.

Thyroidal cAMP-dependent protein kinases. Particular of the type I kinase, and compartmentalization of the two isoenzymes.

Thyroidal cyclic-AMP-dependent protein kinases, present in two subcellular compartments (cytosol and particulate fraction), were characterized using several well established criteria for the identification of the two types of cyclic-AMP-dependent protein kinases (isoenzymes I and 11). By reference to five criteria based on the specific properties of regulatory subunits, thyroidal cytosolic CAMP-dependent protein kinases were identified as type I and type II. This finding is supported by the fact that results for several criteria were identical to those obtained with, commercially available protein kinases type I and II, or protein kinases I and II from other rat tissues (heart, brain) analyzed simultaneously with thyroidal enzymes. It was observed, however, that thyroidal kinase I has some particular qualities which make it distinct from the same type of enzyme in other rat tissues. Thyroidal kinase I has a low sedimentation coefficient (4.9 S) which seems to be an intrinsic property of thyroid cell. The molecular mass of its regulatory subunit (46–48 kDa) corresponds to the weight of the intact native regulatory subunit of kinase I. Another characteristic of thyroidal kinase I is its unusually high molarity of elution (160 mM NaCl), when a low ionic strength buffer is used for homogenization and chromatography. This particular property is specific for the thyroidal enzyme, since it was not observed with rat heart type I kinase. Because of this unusually high molarity of elution of kinase I, the conventional method of protein kinase fractonation by DEAE-cellulose chromatography is not suitable for the separation of thyroidal enzymes. It can be replaced, however, by sucrose gradient ultracentrifugation. Because of the lower sedimentation coefficient of kinase I, the two thyroidal protein kinases can be separated from each other with high yields (80–120%). All results obtained on the particulate protein kinase are consistent, showing that in the particulate extracts only one CAMP-dependent protein kinase entity is present. Its characteristics for the size of the regulatory subunits (46–48 kDa), the molarity of elution from the DEAE-cellulose column, and the value of the sedimentation coefficient are the same as those of the cytosolic type I kinase. We can deduce, therefore, that the particulate protein kinase and the cytosolic type I kinase are the same enzyme entity. A quantitative evaluation showed that the particulate fraction contains about 20%, of the total amount of thyroidal type I kinase. The presence of type I kinase both in the cytosol and in the particulate fraction, and of type II kinase only in the cytosol, lead one to conclude that the two thyroidal CAMP-dependent protein kinases have different subcellular compartmentalization.

Interchain disulfide bonding in the regulatory subunit of cAMP-dependent protein kinase I.

The two protomers of the purified regulatory subunit from porcine cAMP-dependent protein kinase I have been shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into two fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for approximately 15 to 20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all five of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5,400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.

The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II.

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.

Communication between catalytic subunits in hybrid aspartate transcarbamoylase molecules: effect of ligand binding to active chains on the conformation of unliganded, inactive chains.

In the regulatory enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) of Escherichia coli, the six catalytic polypeptide chains are arranged as two distinct catalytic trimers "crosslinked" by three regulatory dimers. Because in allosteric proteins it is assumed that the binding of a ligand to one site promotes a conformational change that affects the subsequent binding to other sites in the oligomeric protein, it was of interest to determine directly whether the effects of ligand binding to chains in one catalytic subunit are "communicated" to unliganded chains in the other subunit. Accordingly, hybrid enzyme molecules were constructed containing sensitive chromophores on the three inactive catalytic chains in one subunit along with an active catalytic subunit and three native regulatory subunits. The derivative exhibited the allosteric properties characteristic of the native enzyme. Communication between the two catalytic subunits was demonstrated by spectral measurements showing that the effects of ligand binding to the three active chains are propagated to the chromophores on the unliganded, inactive chains in the other subunit. Moreover, the change in the tertiary structure of the unliganded catalytic chains is tightly linked to the alteration in the quaternary structure.

Characterization of small cAMP-binding fragments of cAMP-dependent protein kinases.

Monomeric cAMP-binding fragments of molecular mass 16,000 and 14,000 daltons were obtained by Sephadex G-75 chromatography of partially trypsin-hydrolyzed regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively. The Stokes radii were 19.1 and 16.4 A, the frictional ratios were 1.15 and 1.03, and the sedimentation coefficients were 1.94 and 1.91 S for the 16,000- and 14,000-dalton fragments, respectively. The 16,000-dalton fragment retained specific cyclic nucleotide binding characteristics of the native protein. The specificity of cyclic nucleotide binding to the 14,000-dalton fragment (cAMP greater than cIMP = 8-bromo-cAMP = 8-oxo-cAMP greater than cUMP = cGMP) differed from that of the native subunit (cAMP = 8-oxo-cAMP greater than 8-bromo-cAMP greater than cIMP greater than cUMP = cGMP). The 14,000-dalton fragment bound nearly 1 mol of cAMP/mol of fragment. The binding exchange rate of cAMP was much faster for the 14,000-dalton fragment than for either of the native regulatory subunits or for the 16,000 dalton fragment. Although hemin inhibited cAMP binding to the native regulatory subunits and to the 16,000 dalton fragment, the molecule did not affect cAMP binding to the 14,000-dalton fragment. Both of the native regulatory subunits and the isolated 16,000- and 14,000-dalton fragments could be covalently labeled with the photoaffinity analog, 8-N3-[32P]cAMP. The 14,000-dalton fragment could not be phosphorylated and neither fragment could recombine with the catalytic subunit to inhibit its activity. The results indicate that the functional entities of the regulatory subunit other than cAMP binding are destroyed by trypsin. The properties of the 16,000-dalton fragment suggest that the intact cAMP-binding site is contained in a small trypsin-resistant "core" of the native regulatory subunit. The properties of the 14,000-dalton fragment imply that part of the binding site of the native regulatory subunit was slighlty modified or lost during preparation of this fragment.

Correlation of the cAMP binding domain with a site of autophosphorylation on the regulatory subunit of cAMP-dependent protein kinase II from porcine skeletal muscle.

The regulatory subunit of CAMP-dependent protein kinase II from porcine skeletal muscle (subunit M, = 55,000) is labile to specific and limited proteolysis. Both the phosphorylated and dephosphorylated form of the subunit are cleaved by trypsin and chymotrypsin to yield a 37,000 molecular weight CAMP-binding frag- ment and a 16,000 to 17,000 molecular weight fragment. Thermolysin yielded the same proteolytic fragments from the dephosphorylated regulatory subunit; how- ever, the phosphorylated regulatory subunit was rela- tively resistant to proteolysis with thermolysin. Ex- tended proteolysis with trypsin indicated that even smaller CAMP-binding fragments could be generated. If the regulatory subunit was autophosphorylated with [Y-~‘P]ATP or covalently labeled with the photoaf- finity reagent 8-azido-[32P]cAMP, the radioactivity in both cases was retained exclusively with the 37,000 molecular weight fragment following proteolysis with chymotrypsin. This fragment, although retaining the CAMP binding site, was monomeric in comparison to the highly asymmetric dimeric structure of the native regulatory subunit. In contrast to the native regulatory subunit which has a blocked NH2-terminal residue, dansylation of the 37,000 molecular weight fragment gave an NH2-termi- nal aspartic acid, indicating that this fragment corre- sponds to the COOH-terminal end of the native poly- peptide chain. Further sequencing of this fragment yielded the following sequence: Asx-Arg-Arg-Val- P I Ser-Val with 3zP from the autophosphorylated subunit released at Step 5. This sequence indicated that at least one site of autophosphorylation is located near the proteolytically labile site and may explain why the phosphorylated regulatory subunit was resistant to thermolytic cleavage. A model for defining the func- tional domains of the regulatory subunit is presented. Two major forms of CAMP-dependent protein kinases have been identified and are distinguished most frequently by their elution from DEAE-cellulose (l-4). In addition to electrostatic differences, the two types of CAMP-dependent protein kinase, by convention referred to as protein kinase I and II, differ immunologically (5) and genetically (6). Differences are also apparent in the dissociation and reassociation of catalytic and regulatory subunits for the two forms of protein kinase (7-9).

Communication between dissimilar subunits in aspartate transcarbamoylase: Effect of inhibitor and activator on the conformation of the catalytic polypeptide chains

Although local, direct effects of ligand binding to proteins are readily differentiated conceptually from gross, indirect conformational changes in regions of the protein remote from the site of binding, it has been difficult experimentally to distinguish between them. In oligomeric proteins, for example, the binding of ligands to one chain may cause a conformational change in the unliganded chains, but many physical chemical probes are not sufficiently discriminating to demonstrate where the change occurred. Evidence has been lacking as to whether the inhibitor, CTP, or the activator, ATP, in binding to the regulatory chains of the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) causes a conformational change that is propagated throughout the enzyme to the catalytic chains. To demonstrate this "communication" of effects of binding at one site on the conformation of other polypeptide chains, we constructed molecules containing native regulatory subunits and enzymically active nitrated catalytic subunits having one sensitive nitrotyrosyl chromophore per polypeptide chain. These hybrid molecules exhibited the characteristic regulatory properties of the native enzyme. Upon the addition of CTP the population of molecules was shifted toward the constrained or T state, as shown by the change in the sedimentation coefficient and the altered enzyme kinetics. Moreover, there was a decrease in absorbance at 430 nm due to the altered environment of the nitrated catalytic polypeptide chains. In contrast, ATP caused a shift toward the relaxed or R conformation, and the absorbance due to the nitrotyrosyl residues was increased. Different types of experiments indicated that the modified enzyme molecules are in a preexisting equilibrium that is perturbed by CTP or ATP; the resulting conformational changes in the nitrated catalytic subunits are detected by opposite alterations in their absorbance spectrum.

Functionally important arginine residues of aspartate transcarbamylase.

The reaction of phenylglyoxal with aspartate transcarbamylase and its isolated catalytic subunit results in complete loss of enzymatic activity (Kantrowitz, E. R., and Lipscomb, W. N. (1976) J. Biol. Chem. 251, 2688-2695). If N-(phosphonacetyl)-L-aspartate is used to protect the active site, we find that phenylglyoxal causes destruction of the enzyme's susceptibility to activation by ATP and inhibition by CTP. Furthermore, CTP only minimally protects the regulatory site from reaction with this reagent. The modified enzyme still binds CTP although with reduced affinity. After reaction with phenylglyoxal, the native enzyme shows reduced cooperativity. The hybrid with modified regulatory subunits and native catalytic subunits exhibits slight heterotropic or homotropic properties, while the reverse hybrid, with modified catalytic subunits and native regulatory subunits, shows much reduced homotropic properties but practically normal heterotropic interactions. The decrease in the ability of CTP to inhibit the enzyme correlates with the loss of 2 arginine residues/regulatory chain (Mr = 17,000). Under these reaction conditions, 1 arginine residue is also modified on each catalytic chain (Mr = 33,000). Reaction rate studies of p-hydroxymercuribenzoate, with the liganded and unliganded modified enzyme suggest that the reaction with phenylglyoxal locks the enzyme into the liganded conformation. The conformational state of the regulatory subunit is implicated as having a critical role in the expression of the enzyme's heterotropic and homotropic properties.

The nature of the altered allosteric behaviour of 2-thiouracil aspartate transcarbamylase

The aspartate transcarbamylase which has been synthesized by Escherichia coli in the presence of 2-thiouracil has been reported by Kerbiriou & Herve (1972, 1973) to lack homotropic interactions (sigmoidal aspartate saturation kinetics) and yet exhibits heterotropic interactions (CTP inhibition). These observations are of interest in relation to recent theories for the allosteric regulation of ATCase§ which have tried to account for the behaviour of 2-thiouracil ATCase. We have compared the conformational behaviour of 2-thiouracil ATCase with native ATCase, in order to determine the structural basis for the altered allosteric properties. First, measurement of the effects of ligands on the sedimentation coefficient of 2-thiouracil ATCase reveals that the transition from the low-aspartate-affinity (slower sedimenting) form to the high-affinity form occurs more easily with 2-thiouracil ATCase than with native ATCase. Such an effect would be expected to lower co-operativity. Second, measurement of the reactivity of the sulfhydryl groups of 2-thiouracil ATCase indicates that the enzyme reacts faster and is heterogenous with respect to the reactivity of the sulfhydryl groups towards p-hydroxymercuribenzoic acid. This heterogeneity is consistent with the observed heterogeneity of this enzyme on polyacrylamide gels and may complicate the interpretation of kinetic data. Third, the maximal velocity of 2-thiouracil ATCase is approximately half that of native ATCase. Studies using a hybrid of native catalytic subunit and 2-thiouracil ATCase regulatory subunit reveal that the lower maximal velocity is the result of modifications in the catalytic subunit. This suggests that in 2-thiouracil ATCase there may be less than six functional active sites per molecule, an effect which would again lead to lower co-operativity. Finally, kinetic studies at aspartate concentrations lower than previously reported reveal that 2-thiouracil-ATCase is not entirely devoid of co-operativity, exhibiting some residual homotropic and heterotropic interactions.

Cooperative interactions in hybrids of aspartate transcarbamylase containing succinylated regulatory polypeptide chains.

Succinylated derivatives of the regulatory subunit of aspartate transcarbamylase of Escherichia coli were prepared by treating the intact enzyme with succinic anhydride followed by dissociation of the modified protein into catalytic and regulatory subunits which were separated by ion exchange chromatography. The succinylated regulatory subunits were used in hybridization experiments with native subunits to study the organization of the six regulatory polypeptide chains in the intact enzyme. Rapid mixing of succinylated and native regulatory subunits with native catalytic subunits yielded a four-membered hybrid set of reconstituted enzyme-like molecules; hence, the assembly process involves three regulatory combining units and the six regulatory polypeptide chains in the intact enzyme must be arranged as three dimeric subunits. When the modified and native regulatory subunits were incubated together for only brief periods (less than 1 min) followed by the addition of catalytic subunits, the resulting hybrid set was complex with no resolution of discrete species. Apparently the isolated regulatory dimers dissociate readily and reversibly into single polypeptide chains due to relatively weak intra-subunit bonding domains. In contrast, after reconstitution of enzyme-like molecules, the incorporated succinylated regulatory subunits did not exchange with free subunits. Enzyme-like molecules containing three extensively succinylated regulatory subunits show reduced binding of the inhibitor, CTP, and lack both the homotropic and heterotropic effects characteristic of native aspartate transcarbamylase. Preparations containing only slightly succinylated regulatory subunits showed only little inhibition by CTP and considerable cooperativity. The decrease in homotropic effects in these reconstituted molecules correlated with the reduction in the succinate-promoted change in the sedimentation coefficient. Reconstituted enzyme-like molecules containing regulatory subunits which had been extensively succinylated in the presence of CTP retained their binding capacity even though they were only slightly inhibited by CTP and exhibited reduced cooperativity. Hybrid molecules containing both native and succinylated regulatory subunits also possessed reduced allosteric behavior.

Rabbit skeletal muscle protein kinase. Conversion from cAMP dependent to independent form by chemical pertubations

Protein kinase isolated from rabbit skeletal muscle can be reversibly converted from the cAMP dependent form to the indepent form by chaotropic salts and urea. A similar but irreversible conversion can also be induced by trypsin digestion of the holoenzyme. The dissociation of cAMP dependent protein kinase by low concentrations of thiocyanate raises the possibility of isolating both native regulatory and catalytic subunits. From various changes in enzymatic activity caused by urea and trypsin perturbation, it is proposed that the conversion of protein kinase from the cAMP dependent to the independent form is due primarily to preferential modification of the regulatory subunit of the holoenzyme.

Relaxation spectra of aspartate transcarbamylase. I. Interaction of 5-bromocytidine triphosphate with native enzyme and regulatory subunit.

Relaxation spectra of aspartate transcarbamylase. I. Interaction of 5-bromocytidine triphosphate with native enzyme and regulatory subunit.

Hybridization of native and chemically modified enzymes. 3. The catalytic subunits of aspartate transcarbamylase.

Succinylation of the catalytic subunits of ATCase yielded a relatively homogeneous, inactive electrophoretic variant which upon mixing with native regulatory subunits formed a complex the size of the native enzyme. Hybridization experiments with mixtures of this variant and the native catalytic subunits in the presence of excess regulatory subunits yielded three different molecular complexes which were separated and individually characterized. The number and properties of the various components indicated that each ATCase molecule contains two catalytic subunits. Hybridization was also effected at the intrasubunit level by dissociation and reconstitution of mixtures of the native and modified catalytic subunits. These experiments produced four components showing thereby that each catalytic subunit is composed of three polypeptide chains. The potential use of the various hybrids is discussed in relation to the unique properties manifested by regulatory enzymes.

Immunological studies of aspartate transcarbamylase. I. Characterization of the native enzyme, catalytic, and regulatory subunit immune systems.

Immunological studies of aspartate transcarbamylase. I. Characterization of the native enzyme, catalytic, and regulatory subunit immune systems.