ABSTRACT
Phosphorylase kinase (PhK), a 1.3 MDa enzyme complex that regulates glycogenolysis, is composed of four copies each of four distinct subunits (α, ß, γ, and δ). The catalytic protein kinase subunit within this complex is γ, and its activity is regulated by the three remaining subunits, which are targeted by allosteric activators from neuronal, metabolic, and hormonal signaling pathways. The regulation of activity of the PhK complex from skeletal muscle has been studied extensively; however, considerably less is known about the interactions among its subunits, particularly within the non-activated versus activated forms of the complex. Here, nanoelectrospray mass spectrometry and partial denaturation were used to disrupt PhK, and subunit dissociation patterns of non-activated and phospho-activated (autophosphorylation) conformers were compared. In so doing, we have established a network of subunit contacts that complements and extends prior evidence of subunit interactions obtained from chemical crosslinking, and these subunit interactions have been modeled for both conformers within the context of a known three-dimensional structure of PhK solved by cryoelectron microscopy. Our analyses show that the network of contacts among subunits differs significantly between the nonactivated and phospho-activated conformers of PhK, with the latter revealing new interprotomeric contact patterns for the ß subunit, the predominant subunit responsible for PhK's activation by phosphorylation. Partial disruption of the phosphorylated conformer yields several novel subcomplexes containing multiple ß subunits, arguing for their self-association within the activated complex. Evidence for the theoretical αßγδ protomeric subcomplex, which has been sought but not previously observed, was also derived from the phospho-activated complex. In addition to changes in subunit interaction patterns upon phospho-activation, mass spectrometry revealed a large change in the overall stability of the complex, with the phospho-activated conformer being more labile, in concordance with previous hypotheses on the mechanism of allosteric activation of PhK through perturbation of its inhibitory quaternary structure.
Subject(s)
Catalytic Domain , Muscle, Skeletal/enzymology , Phosphorylase Kinase , Protein Subunits/analysis , Catalysis , Mass Spectrometry , Muscle, Skeletal/metabolism , Phosphorylase Kinase/analysis , Phosphorylase Kinase/chemistry , Phosphorylase Kinase/metabolism , Phosphorylation , Protein Conformation , Protein Structure, Quaternary , Protein Subunits/chemistryABSTRACT
Phosphorylase kinase (PhK) is a large hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase (GP). It consists in four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha beta delta). Delta corresponds to endogenous calmodulin, whereas little is known on the molecular architecture of the large alpha and beta subunits, which probably arose from gene duplication. Here, using sensitive methods of sequence analysis, we show that the C-terminal domain (named domain D) of these alpha and beta subunits can be significantly related to calcineurin B-like (CBL) proteins. CBL are members of the EF-hand family that are involved in the regulation of plant-specific kinases of the CIPK/PKS family, and relieve autoinhibition of their target kinases by binding to their regulatory region. The relationship highlighted here suggests that PhK alpha and/or beta domain D may be involved in a similar regulation mechanism, a hypothesis which is supported by the experimental observation of a direct interaction between domain D of PhKalpha and the regulatory region of the Gamma subunit. This finding, together the identification of significant similarities of domain D with the preceding domain C, may help to understand the molecular mechanism by which PhK alpha and/or beta domain D might regulate PhK activity.
Subject(s)
Calcineurin/chemistry , Phosphorylase Kinase/chemistry , Phosphorylase Kinase/metabolism , Protein Structure, Tertiary , Amino Acid Sequence , Animals , Calcineurin/analysis , Calcineurin/metabolism , Calcium/metabolism , Calmodulin/metabolism , Cluster Analysis , Conserved Sequence , Databases, Factual , EF Hand Motifs , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Phosphorylase Kinase/analysis , Phosphorylase Kinase/genetics , Phosphorylase Kinase/physiology , Protein Binding , Protein Folding , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits/analysis , Protein Subunits/chemistry , Protein Subunits/metabolism , Rabbits , Sequence Analysis, Protein , Sequence Homology, Amino AcidABSTRACT
Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.
Subject(s)
Allosteric Regulation/drug effects , Allosteric Site/drug effects , Cross-Linking Reagents/pharmacology , Mass Spectrometry/methods , Peptides/metabolism , Phosphorylase Kinase/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Animals , Models, Biological , Molecular Sequence Data , Mutant Proteins/analysis , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Phosphorylase Kinase/analysis , Phosphorylase Kinase/chemistry , Phosphorylation/drug effects , Phosphoserine/metabolism , Point Mutation/genetics , Protein Binding/drug effects , Protein Interaction Mapping , Protein Structure, Quaternary/drug effects , Protein Structure, Tertiary/drug effects , Protein Subunits/analysis , Protein Subunits/chemistry , Protein Subunits/metabolism , Rabbits , Sequence Deletion/genetics , Structural Homology, Protein , Succinimides/pharmacologyABSTRACT
Raf-1 is a serine/threonine protein kinase that plays a critical role in mitogenic signal transduction. Raf-1 activation requires 14-3-3 binding to Raf-1 as an essential step. This binding is regulated through phosphorylation of Ser259 and Ser621 of Raf-1, each constituting part of the consensus motif for the binding of Raf-1 to 14-3-3. However, Raf-1 kinase kinase(s) that phosphorylates these sites remains unknown. In this report, we detected Raf-1 kinase kinase activity using recombinant glutathione-S-transferase-Raf-1 fusion proteins as substrate of in situ gel kinase assay. Ser259 was phosphorylated by a kinase with a molecular weight of 90 kDa, which was suggested to be Rsk judging from the molecular size, the time course of activation after EGF stimulation and the elution pattern from an anion-exchange column. The Raf-1 fragment containing Ser621 was phosphorylated by kinases with molecular weights of 85, 60, 50 and 48 kDa but not by the kinase that phosphorylates Ser259. These results suggest that although Ser259 and Ser621 lie in the same amino acid sequence motif for 14-3-3 binding, these two regulatory sites for this binding are phosphorylated by different protein kinases.
Subject(s)
Phosphorylase Kinase/analysis , Proto-Oncogene Proteins c-raf/metabolism , Animals , Autoradiography , Binding Sites , Cells, Cultured , Epidermal Growth Factor/pharmacology , Gels , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , Mice , Mutation/physiology , Proto-Oncogene Proteins c-raf/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/drug effects , Serine/metabolismABSTRACT
The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.
Subject(s)
Astrocytes/enzymology , Brain/cytology , Brain/enzymology , Neurons/enzymology , Phosphorylase Kinase/analysis , Animals , Astrocytes/cytology , Cells, Cultured , Fluorescent Antibody Technique , Immunoenzyme Techniques , Male , Neurons/cytology , Rats , Rats, WistarABSTRACT
A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylase a, i.e., glycogen and glucose 1-phosphate (or P(i)), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase.
Subject(s)
Phosphorylase Kinase/analysis , Animals , Enzyme Activation , Glycogen/analysis , Kinetics , Muscle, Skeletal/enzymology , Nephelometry and Turbidimetry , Phosphorus Radioisotopes , Phosphorylase Kinase/chemistry , Phosphorylase a/chemistry , Phosphorylase b/chemistry , RabbitsABSTRACT
The evaluation of glycogen phosphorylase kinase in rat brain subcellular fractions was undertaken in order to get further insight into the association of this kinase with specific neuronal cell compartments. The enzyme was found to be primarily soluble, but considerable latent specific activities were observed in particulate fractions, especially in microsomes, mitochondria and synaptosomes, which could be unmasked by treatment with Triton-X-100. The submitochondrial and subsynaptic distribution patterns of phosphorylase kinase revealed high overt activity in the mitochondrial intermembrane space and high latent activities in mitochondrial membranes, and synaptic vesicles, membranes and mitochondria. The Ca(2+)-dependency of soluble phosphorylase kinase was similar to that of microsomal enzyme but higher than that of other particulate enzyme forms. Mitochondrial phosphorylase kinase showed a higher pH 6.8:8.2 activity ratio than the soluble and the microsomal enzyme. The rate of inactivation of cytosolic phosphorylase kinase by proteinase K was higher than that of microsomal and mitochondrial enzymes. Antibodies against rabbit skeletal muscle phosphorylase kinase effectively inhibited both cytosolic and microsomal enzyme but failed to significantly affect the kinase activity present in intact mitochondria and intermembrane space. Western blotting with anti-phosphorylase kinase showed that rat brain mitochondria exhibited a significantly lower immunoreactivity compared to soluble cytosol. In conclusion, the presence of phosphorylase kinase activity in a variety of particulate fractions of rat brain suggests a multiplicity of actions of this kinase in neuronal tissues.
Subject(s)
Brain/enzymology , Mitochondria/enzymology , Neurons/enzymology , Phosphorylase Kinase/analysis , Subcellular Fractions/enzymology , Animals , Brain/ultrastructure , Calcium/pharmacology , Cytosol/enzymology , Hydrogen-Ion Concentration , Hydrolysis , Immunoblotting , Isoenzymes/immunology , Membranes/enzymology , Neurons/ultrastructure , Phosphorylase Kinase/antagonists & inhibitors , Phosphorylase Kinase/immunology , RatsABSTRACT
A continuous spectrophotometric assay for the determination of the initial rate of the phosphorylase kinase catalyzed reaction at pH 7.0 is presented. The assay incorporates two coupling enzyme systems: (a) recombinant rabbit skeletal muscle type 1 protein phosphatase catalytic subunit which dephosphorylates the phosphorylase a product of the phosphorylase kinase reaction, and (b) the system of Webb (Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992), which uses purine nucleoside phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for the quantitation of the resultant inorganic phosphate. The effects of reaction components on the enzyme activities were studied. The system was standardized and validated. The continuous coupled enzyme system was used for the kinetic analysis of nonactivated phosphorylase kinase at pH 7.0. Km and kcat values of 15.36 +/- 0.2 microM (phosphorylase b monomer) and 21 +/- 1.12 s-1, respectively, were determined.
Subject(s)
Phosphoprotein Phosphatases/analysis , Phosphorylase Kinase/analysis , Spectrophotometry/methods , Animals , Binding Sites , Catalysis , Kinetics , Phosphorylases/chemistry , Purine-Nucleoside Phosphorylase/chemistry , Rabbits , Regression Analysis , Reproducibility of ResultsABSTRACT
To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Cyclic AMP-Dependent Protein Kinases/analysis , Glycogen/metabolism , Oncorhynchus mykiss/physiology , Phosphorylase Kinase/analysis , Physical Exertion/physiology , Animals , Cyclic AMP/analysis , Enzyme Activation , Liver/enzymology , Liver/metabolism , Muscles/enzymology , Muscles/metabolism , Phosphorylases/metabolism , Second Messenger Systems , Swimming , Tissue DistributionABSTRACT
Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.
Subject(s)
Adenosine Triphosphate/analysis , Fluoresceins , Phosphorylase Kinase/analysis , Thiocyanates , Adenosine Triphosphate/pharmacology , Affinity Labels , Amino Acid Sequence , Amino Acids/analysis , Binding Sites , Binding, Competitive , Fluorescein-5-isothiocyanate , Fluoresceins/pharmacology , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Sequence Data , Peptide Fragments/analysis , Phosphorylase Kinase/antagonists & inhibitors , Thiocyanates/pharmacologyABSTRACT
Using DEAE-Toyopearl column chromatography, a preparation of pigeon skeletal muscle phosphorylase kinase was obtained in a state approaching homogeneity. The molecular mass of the native enzyme (1320 kDa) and the subunit formula (alpha beta gamma delta)4 are similar to those of rabbit and chicken counterparts. Both red and white pigeon skeletal muscle isozymes contain the alpha'-subunit instead of alpha. Gradient SDS-PAGE electrophoresis revealed small but well-reproducible differences in the molecular masses of rabbit, chicken and pigeon muscle beta- and gamma-subunits. The activity ratio at pH 6.8/8.2 is 0.06-0.15 for different preparations of phosphorylase kinase b. The activity of pigeon muscle phosphorylase kinase b is Ca2+-dependent. The [Ca2+]0.5 value at pH 7.0 is 20 microM, which exceeds that for the chicken muscle enzyme by two orders of magnitude. In the presence of Ca2+, pigeon phosphorylase kinase b is activated 4-fold by saturating concentrations of calmodulin and troponin C. Pigeon muscle phosphorylase b is activated 3-5-fold during autophosphorylation or phosphorylation by the catalytic subunit of cAMP-dependent protein kinase.
Subject(s)
Columbidae/metabolism , Muscles/enzymology , Phosphorylase Kinase/isolation & purification , Animals , Calcium/pharmacology , Calmodulin/physiology , Chickens , Enzyme Activation/drug effects , Macromolecular Substances , Phosphorylase Kinase/analysis , Phosphorylation , Rabbits , Troponin/physiology , Troponin CABSTRACT
A sample of phosphorylase kinase aged by storage was subjected to chromatography on a size exclusion column. Samples from the four major peaks were analysed by electron microscopy. The first major peak consisted of oligomers of the enzyme monomers which showed no structural order. The second peak mainly consisted of dimers of phosphorylase kinase connected to each other by the outer tipes of the "wings", thus identifying a binding locus through cross-labelling. The third peak showed the already known typical forms of phosphorylase kinase ("butterflies", "chalice form"). The last peak yielded structures identical to the alpha gamma delta fragments obtained by lithium bromide cleavage of the intact enzyme. These structures correspond to the curved outer parts of the cup substructures of the chalice and butterfly forms. The result could be verified by computer averaging of the alpha gamma delta fragments. This finding confirms reports (Chan, K.-F. J. & Graves, D.J. (1984) Calcium Cell Funct. 5, 1-31) that the beta-subunits (missing in the distally situated fragment) are located in the central parts of the molecules. The good quality of the averages of the low molecular mass fragment (about 200 kDa) recommends computer averaging of electron micrographs of partial complexes as suitable method for the study of protein complexes.
Subject(s)
Lithium Compounds , Phosphorylase Kinase/analysis , Bromides , Electrophoresis, Polyacrylamide Gel , Lithium , Microscopy, Electron , Peptide Fragments/analysis , Protein ConformationABSTRACT
We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.
Subject(s)
Cloning, Molecular , DNA/analysis , Phosphorylase Kinase/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calmodulin/metabolism , Macromolecular Substances , Methionine , Molecular Sequence Data , Molecular Weight , Phosphorylase Kinase/analysis , Protein Processing, Post-Translational , RabbitsABSTRACT
We have isolated and sequenced a cDNA encoding the alpha subunit of phosphorylase kinase from rabbit fast-twitch skeletal muscle. The cDNA molecule consists of 388 nucleotides of 5'-nontranslated sequence, the complete coding sequence of 3711 nucleotides, and 342 nucleotides of 3'-nontranslated sequence followed by a poly(dA) tract. It encodes a polypeptide of 1237 amino acids and a deduced molecular mass of 138,422 Da. Nearly half of the deduced amino acid sequence is confirmed by peptide sequencing. Seven positions of endogenously phosphorylated serine residues and autophosphorylation sites, identified by peptide sequencing, could be assigned. They cluster in a segment of only 60 amino acids. RNA blot hybridization analysis demonstrates a predominant RNA species of approximately equal to 4500 nucleotides and a less abundant RNA of 8700 nucleotides.
Subject(s)
Cloning, Molecular , DNA/analysis , Muscles/enzymology , Phosphorylase Kinase/genetics , Amino Acid Sequence , Animals , Base Sequence , Molecular Sequence Data , Molecular Weight , Phosphorylase Kinase/analysis , Poly A/analysis , RNA, Messenger/analysis , RabbitsABSTRACT
Molecular structures related to phosphorylase kinase have been localized by light and electron microscopy in tissue sections of rabbit skeletal muscle employing polyclonal antibodies directed against the holoenzyme as well as monoclonal antibodies specific for its alpha-, beta- or gamma-subunits. In frozen sections of prefixed muscle fibres both known major regions of glycogen deposition, the intermyofibrillar space and the perinuclear area, are stained predominantly. In sections of unfixed muscle in which cytosolic phosphorylase kinase was removed by extensive washes prior to immunostaining the immunolabel is mainly associated with the sarcoplasmic reticulum (SR). This membrane location is further confirmed by immunoblot analysis of proteins solubilized from isolated SR with Triton X-114. Employing monoclonal antibodies two membrane proteins are identified as the alpha- and beta-subunits of phosphorylase kinase by Western blots. Immunoprecipitates reveal also the gamma-subunit; the delta-subunit, i.e., calmodulin, is enriched with the solubilized enzyme. It proves that a SR membrane associated form of holophosphorylase kinase exists in muscle. Functionally, this kinase might be involved in phosphorylation of phosphatidylinositol present on the SR Ca2+ transport ATPase and thereby might play a role in regulation of Ca2+ transport.
Subject(s)
Muscles/enzymology , Phosphorylase Kinase/analysis , Sarcoplasmic Reticulum/enzymology , Animals , Antibodies, Monoclonal , Centrifugation, Density Gradient , Fluorescent Antibody Technique , Immunoassay , Immunohistochemistry , Microscopy, Electron , Microsomes/enzymology , Muscles/ultrastructure , Phosphorylase Kinase/immunology , Rabbits , Sarcoplasmic Reticulum/ultrastructureABSTRACT
Synthetic oligonucleotides have been used to isolate a 1.85 kb clone containing the full length coding sequence for the catalytic subunit of rabbit skeletal muscle phosphorylase kinase from a cDNA library constructed in lambda gt10. Sequence analysis of the clone predicted an amino acid sequence in agreement with a published primary structure. Inspection of the codon usage revealed a strong preference for G or C nucleotides at the third codon position as found for several other skeletal muscle proteins. This cDNA clone should facilitate identification of functional domains, including the calmodulin-binding site, and investigation of the molecular basis of X-linked phosphorylase kinase deficiencies.
Subject(s)
DNA/isolation & purification , Phosphorylase Kinase/genetics , Amino Acid Sequence , Animals , Base Sequence , Catalysis , Cloning, Molecular , Codon , Muscles/enzymology , Phosphorylase Kinase/analysis , RabbitsABSTRACT
The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles. The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.
Subject(s)
Glycogen/analysis , Hair/metabolism , Phosphorylase Kinase/analysis , Phosphorylase a/analysis , Phosphorylase b/analysis , Phosphorylases/analysis , Cells, Cultured , DNA/analysis , Hair/enzymology , Humans , MicrochemistryABSTRACT
Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.