Rajendra K. Sharma

N-myristoyl transferase assay using phosphocellulose paper binding

N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.

Biochemical and biophysical characterization of the reovirus cell attachment protein σ1 : evidence that it is a homotrimer

Abstract The oligomerization state of the reovirus cell attachment protein σ1 (49K monomeric molecular weight) was determined by biochemical and biophysical means. Full-length (protein product designated A) and C-terminal truncated (protein product designated B) serotype 3 reovirus Sl mRNA transcripts synthesizedin vitro were cotranslated in a rabbit reticulocyte lysate, and the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under conditions which allowed for the identification of oligomeric forms of σl. A total of four oligomeric protein bands (corresponding to A3, A2B1, A1B2, and B3, respectively) was consistently observed, which suggests that the protein is made up of three monomeric subunits. Biophysical characterization of purified σ1 using column filtration and sucrose gradient sedimentation analysis confirmed the highly asymmetric shape of σ1 and allowed us to determine the molecular weight of the native protein to be ∼132K (a trimer). Similar biophysical analysis on the two tryptic fragments of the σ1 [N-terminal fibrous tail (26K monomeric molecular weight) and the C-terminal globular head (23K monomeric molecular weight)] yielded molecular weights of 77K and 64K, respectively, both again corresponding to trimers. We therefore conclude that protein σ1 is a homotrimer and provide, with supportive experimental evidence, a rationale for the anomalous behavior of the oligomeric protein in SDS-polyacrylamide gels, which, coupled with chemical cross-linking studies, has in part led to the previous suggestion that σ1 might be a higher order oligomer.

The differential stimulation of brain and heart cyclic-AMP phosphodiesterase by oncomodulin

Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, from the bovine heart and brain, purified by monoclonal antibody chromatography were tested with respect to activation by oncomodulin. The heart and brain enzymes which have previously been shown to have slightly different electrophoretic mobilities (1), were found to also differ in the oncomodulin dose-dependent activation of cAMP hydrolysis. Oncomodulin was shown to activate the heart enzyme to the same extent as calmodulin. However, this study indicates that the heart phosphodiesterase has approximately 25-fold higher affinity for oncomodulin than the brain enzyme. The oncomodulin concentration required for the half-maximal activation of the heart phosphodiesterase was estimated to be 2 X 10(-7)M. In addition, the possibility of the observed activation by oncomodulin being due to calmodulin contamination can be ruled out as the oncomodulin activation profiles were unaltered subsequent to chromatography on organomercurial agarose and the activation by oncomodulin could not be reversed by anti-calmodulin IgG.

The Calmodulin Regulatory System

Publisher Summary This chapter focuses on the calmoudulin regulatory system. All cell types contain an elaborate regulatory network that permits the cell to respond to a variety of external and internal stimuli. The metabolism and the mechanism of action of the second messengers are under rigorous and complex control with the participation of numerous enzymes and regulatory proteins. Calmodulin is one of the most catholic regulators and is involved in control of both cAMP- and Ca2+-mediated systems. Calmodulin was discovered as an activator of cyclic nucleotide phosphodiesterase by Cheung and demonstrated to play a role in Ca 2+ regulation of the enzyme by Kakiuchi and Yamazaki. The pivotal role played by calmodulin in the second messenger system is because of several unique features of this protein. The identification of calmodulin-regulated enzymes has been made by in vitro testing of the Ca 2+ -dependent calmodulin binding to and modulation of the activity of enzymes of known biological function. In this way, calmodulin has been demonstrated to regulate enzymes involved in glycogen metabolism, cyclic nucleotide metabolism, calcium metabolism, and contractile processes. In most cases, the regulation of calmodulin-dependent processes is initiated by binding of Ca 2+ to free calmodulin to convert the protein from an inactive to an active conformation. The activated calmodulin associates with the target enzymes to modulate the latters activity.

Purification and characterization of the heat-stable calmodulin-binding protein from the matrix of bovine heart mitochondria

A heat-stable calmodulin binding protein was purified and characterized from the matrix of bovine heart mitochondria. It bound specifically to calmodulin in the presence of calcium, and strongly inhibited the stimulatory activity of calmodulin on phosphodiesterase. The estimated molecular weight of the calmodulin-binding protein was 61,000 dalton determined by SDS-polyacrylamide gel electrophoresis.

Mechanisms of Inhibition of Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterase by Dihydropyridine Calcium Antagonists

Abstract: Calmodulin‐dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interaction between the cyclic nucleotide and Ca2+ second‐messenger systems. CaMPDE exists as tissue‐specific isozymes, and initially these isozymes were designated according to their respective subunit molecular mass. A variety of pharmacological agents have been used to inhibit CaMPDE, and this inhibition occurs mostly via Ca2+‐dependent association with the proteins. We have examined the effect of dihydropyridine Ca2+‐channel blockers felodipine and nicardipine on CaMPDE. The results suggest that the 63‐kDa (PDE 1B1) and 60‐kDa (PDE 1A2) CaMPDE isozymes are inhibited by felodipine and nicardipine by partial competitive inhibition and that these two Ca2+ antagonists appear to counteract each other. This study further demonstrates the existence of a specific site, distinct from the active site on CaMPDE, that exhibits high‐affinity binding of these drugs. Felodipine and nicardipine have similar affinities for 60‐kDa CaMPDE isozymes but bring about different levels of enzyme inhibition, suggesting the possibility of designing specific drugs that can protect the enzyme from inhibition by dihydropyridine Ca2+‐channel blockers.

Inhibition of bovine brain calmodulin-dependent cGMP phosphodiesterase by peptide and non-peptide angiotensin receptor ligands

Inhibition of a purified 60 KDa bovine brain calmodulin-dependent cGMP phosphodiesterase (PDE) was investigated for a number of peptides and non-peptides which are known to bind to angiotensin (ANG) receptors. The peptide antagonists sarilesin and sarmesin had KI = 120 and greater than 200 microM respectively, and the peptide agonists ANG II and ANG III had KI = greater than 200 and 45 microM respectively. Non-peptide ANG receptor antagonists related to DuP 753 exhibited KI values in the same range. For both peptide and non-peptide antagonists, inhibitory activities in the PDE assay reflected the order of antagonist potencies at ANG receptors in the rat isolated uterus assay and binding affinities at ANG receptors in rat uterine membranes, suggesting that molecular recognition factors are similar for both ANG receptors and cGMP PDE. The vasodilatory and blood pressure lowering effects of compounds related to DuP 753 may be due in part to inhibition of cGMP PDE. The differential effects of ANG II and ANG III at target tissues may relate in part to the marked differences in cGMP PDE inhibition associated with these two peptides hormones.

[54] Isolation of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes

Publisher Summary This chapter describes the procedures for the demonstration and isolation of bovine brain calmodulin (CaM)-dependent phosphodiesterase isozymes. Purified bovine brain CaM-dependent cyclic nucleotide phosphodiesterase exhibits essentially a single protein band on neutral SDS-PAGE gel but shows two protein bands of apparent M r 60,000 and 63,000 on alkaline SDS-PAGE gel. Using this enzyme preparation as the antigen, seven antiphosphodiesterase monoclonal antibodies have been produced and purified. Western immunoblotting analysis has shown that these antibodies may be divided into two groups, one specific toward the 60-kDa polypeptide and the other reacting with both 60-kDa and the 63-kDa polypeptides. A procedure is developed to use these antibodies to demonstrate that the polypeptides represent subunits of different phosphodiesterase isozymes. The chapter summarizes some of the kinetic and regulatory properties of the isolated CaM-dependent cyclic nucleotide phosphodiesterase isozymes from bovine brain. Both isozymes can rise either cAMP or cGMP as substrates and both exhibit higher affinity for cGMP than for cAMP.

Regulation of the 63-kDa Subunit-Containing Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase Isozyme

Calmodulin-stimulated cyclic nucleotide phosphodiesterase, originally thought to be a single enzyme species in all tissues has been shown to exist in isozymic forms [for review, 1–5]. Homogeneous preparations of calmodulin-dependent phosphodiesterases showing distinct kinetic, regulatory, molecular and immunological properties have been obtained [for review, see (1)]. The well-characterized mammalian isozymes possess low basal (Ca2+-independent) activity and exhibit relatively low affinity towards cyclic AMP. Thus, it appears that these enzymes function most efficiently when cells are stimulated to increase the concentration of both Ca2+ and cAMP and, as such, they are expected to play important roles in integrating the regulatory actions of Ca2+ and cAMP.

[41] Monoclonal antibodies as specific probes for the study of CaM-regulatory systems

Publisher Summary The development of monoclonal antibody hybridoma technology has greatly enhanced the power of immunological approaches in biomedical research. Among other applications, monoclonal antibodies are used as highly specific reagents to probe structure-function relationships in proteins, to study the function of membrane proteins in their native environment, and to purify proteins existing in minute quantities in the cell and to separate closely related proteins. The CaM-regulatory system, with its many unique features, is found to be an area where the application of monoclonal antibodies is especially productive. This chapter is illustrated with four examples that show the versatility of monoclonal antibodies in the study of the CaM-regulatory system. The cross-reactive antibodies are used to identify the common structures existing in this family of proteins. The phosphodiesterase isozyme specific monoclonal antibodies show the potential of the antibody in differentiating and separating structurally very similar proteins. The use of monoclonal antibodies to detect different conformations of an enzyme is illustrated by the conformation specific anti-calcineurin antibody. As CaM-regulated enzymes include protein kinases and phosphatases, CaM can exert its regulatory action indirectly through these enzymes. The last example that describes phospholamban antibodies illustrates the application of monoclonal antibodies to the study of proteins that are indirectly regulated by CaM.