Thomas C. Vanaman

Identification of the Ca2+‐dependent modulator protein as the fourth subunit of rabbit skeletal muscle phosphorylase kinase

Kakuichi et al. [l] and Cheung [2,3] were the first to demonstrate the presence of a factor in brain homogenates, which in the presence of Ca”‘, stimulated the activity of one of the cyclic nucleotide phosphodiesterases of this tissue. This factor was subsequently shown to be a small heat stable calcium binding protein, which was present in high concentrations in a wide variety of animal tissues [4-61. Following its purification to apparent homogeneity from bovine brain [7] and bovine heart [8] , it was noted that its physico-chemical properties were very similar to the calcium-binding subunit of rabbit skeletal muscle troponin, troponln-C, the protein which confers calcium sensitivity to actomyosin ATPase [9,10] . This idea was substantiated by the determination of the amino acid sequence of the ‘calcium-dependent modulator’ from bovine brain [ 1 l] and rat testis [ 121, which showed extensive homology with troponin-C, and by the finding that the ‘modulator’ could substitute for troponin-C in restoring calcium sensitivity to actomyosin ATPase in reconstituted systems [ 131. Troponin-C can also substitute for the ‘modulator’ in the activation of cyclic nucleotide

A possible three-dimensional structure of bovine α-lactalbumin based on that of hen's egg-white lysozyme

Abstract Bovine α-lactalbumin and hen egg-white lysozyme have closely similar amino acid sequences. A model of α-lactalbumin has been constructed on the basis of the main chain conformation established for lysozyme. The side chain interactions of lysozyme are listed (Table 2) and the consequences of the side chain replacements in α-lactalbumin examined. Changes in internal side chains are generally interrelated in a convincing manner, suggesting that the model is largely correct, but there are some regions where it has not been possible to deduce the conformation unequivocally. Glu 35, which acts as a proton donor in lysozyme, is absent in α-lactalbumin, in which a neighbouring histidine residue may assume a similar function. The surface cleft, which is the site of substrate binding in lysozyme, is shorter in α-lactalbumin. While this would be consistent with α-lactalbumin having a mono- or disaccharide as substrate, the biochemical evidence shows that the role of α-lactalbumin in the synthesis of lactose is a complex one requiring direct interaction with the A protein.

Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine

Abstract The reversible, calcium-dependent binding of a calmodulin to phenothiazines has been demonstrated using an immobilized chlorpromazine analog. Calmodulin has been purified from crude extracts of bovine brain utilizing calcium-dependent binding to phenothiazine-Sepharose 4B as an initial affinity-based chromatographic procedure. Chromatography of a crude extract of bovine brain, prepared under non-denaturing conditions, yielded calmodulin contaminated with several other minor EGTA-elutable components. These components were removed by calcium-dependent affinity chromatography on calmodulin-Sepharose 4B and ion-exchange chromatography.

Effect of the troponin C-like protein from bovine brain (brain modulator protein) on the Mg2+-stimulated ATPase of skeletal muscle actomyosin

Increasing evidence indicates that the divalent cation Ca”and the cyclic nucleotides act as central regulatory signals for numerous events in animal cells (for review, see [l-3] ). In vertebrate muscle, Ca2+ regulates actomyosin activity primarily through its action on the calcium binding component of the troponin complex, troponin C. Recently, a ubiquitous troponin C-like protein with high affinity for Ca2’ has been shown to be present in relatively high levels as a soluble component in neurosecretory tissue of numerous vertebrate species [4] and in rapidly proliferating frbroblasts [5,6]. This protein, referred to as modulator protein*, is structurally similar to vertebrate muscle troponin C [7], and has previously been shown to possess two potential regulatory functions in animal cells. First, in the presence of calcium this protein will stimulate the activity of partially purified preparations of cyclic nucleotide phosphodiesterase as first shown by Cheung [8] and Kakiuchi [9]. Second, Cheung and co-workers [ IO,1 1 ] and others [ 121 have shown that modulator protein is also a calcium-dependent stimulator of partially purified preparations of Lubrol solubilized adenyl cyclase activity. We report here that modulator protein possesses troponin C-like activity in Ca2+-regulated actomyosin systems from skeletal muscle. In the presence of troponin I and tropomyosin from skeleta muscle it is as effective in

Regulation of Platelet Plasma Membrane Ca2+-ATPase by cAMP-dependent and Tyrosine Phosphorylation

As a consequence of its central role in the regulation of calcium metabolism in the platelet, the plasma membrane Ca2+-ATPase (PMCA) was assessed for cAMP-dependent and tyrosine phosphorylation. Addition of forskolin or prostaglandin E1, agents known to elevate platelet cAMP and calcium efflux, to platelets pre-labeled with [32P]PO4 resulted in the direct phosphorylation of platelet PMCA. Similarly, addition of the catalytic subunit of protein kinase A to platelet plasma membranes resulted in a 1.4-fold stimulation of activity. Thus, the previously reported inhibition of platelet activation by elevated intracellular cAMP may be accomplished in part by stimulation of PMCA, likely resulting in a decrease in intracellular calcium. Treatment with thrombin evoked tyrosine phosphorylation of platelet PMCA, while PMCA from resting platelets exhibited little tyrosine phosphorylation. Phosphorylation of platelet plasma membranes by pp60 src resulted in 75% inhibition of PMCA activity within 15 min. Similarly, membranes isolated from thrombin-treated platelets exhibited 40% lower PMCA activity than those from resting platelets. Phosphorylation of erythrocyte ghosts and purified PMCA by pp60 src also resulted in up to 75% inhibition of Ca2+-ATPase activity, and inhibition was correlated with tyrosine phosphorylation. Sequencing of a peptide obtained after32P labeling of purified erythrocyte PMCA in vitro showed that tyrosine 1176 of PMCA4b is phosphorylated by pp60 src . These results indicate that tyrosine phosphorylation of platelet PMCA may serve as positive feedback to inhibit PMCA and increase intracellular calcium during platelet activation.

Affinity chromatography purification of a cyclic nucleotide phosphodiesterase using immobilized modulator protein, a troponin C-like protein from brain

Abstract Modulator protein, a brain troponin C-like protein, has been coupled to Sepharose 4B using conditions that allow retention of phosphodiesterase stimulatory activity. This conjugate has been used to directly demonstrate the calcium dependent formation of a reversible modulator protein-phosphodiesterase complex and to purify a cyclic nucleotide phosphodiesterase by affinity chromatography.

Presence of calmodulin in Tetrahymena

Ca-dependent affinity chromatography on phenothiazine-Sepharose 4B has been used to isolate a pure protein from the ciliate Tetrahymena pyriformis. This protein has been identified as calmodulin by demonstrating three of the Ca-dependent activities attributed to calmodulins. Tetrahymena calmodulin also has physicochemical properties similar to those of the previously characterized mammalian, coelenterate, and plant proteins, except for a lower molecular weight (15,000) and slightly different CNBr fragments compared to bovine brain calmodulin. Calmodulin is a constituent of demembranated Tetrahymena cilia from which it can be extracted with the crude dynein fraction. Sucrose density gradient fractionation indicated its presence in fractions containing the 14S dynein ATPase. It is concluded that the essential properties of calmodulin have been highly conserved during much of eukaryotic evolution, and it is suggested that calmodulin plays a role in the control of ciliary motility in Tetrahymena.

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

Critical to SNARE protein function in neurotransmission are the accessory proteins, solubleN-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. In this report, we demonstrate the depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitrophosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF.

In vitro enzyme activation with calbindin‐D28k, the vitamin D‐dependent 28 kDa calcium binding protein

Purified porcine erythrocyte membrane Ca2+‐ATPase and 3′:5′‐cyclic nucleotide phosphodiesterase were stimulated in a dose‐dependent, saturable manner with the vitamin D‐dependent calcium binding protein from rat kidney, calbindin‐D28k (CaBP‐D28k). The concentration of CaBP‐D28k required for half‐maximal activation (K 0.5 act.) of the Ca2+‐ATPase was 28 nM compared to 2.2 nM for calmodulin (CaM), with maximal activation equivalent upon addition or either excess CaM or CaBP‐D28k, 3′:5′‐Cyclic nucleotide phosphodiesterase (PDE) also showed equivalent maximum saturable activation by calbindin (K 0.5 act. = 90 nM) or calmodulin (K 0.5 act. = 1.2 nM). CaBP‐D28k was shown to effectively compete with CaM‐Sepharose for PDE binding. Immunoprecipitation with CaBP‐D28k antiserum completely inhibited calbindin‐mediated activation of PDE but had no effect on calmodulins ability to activate PDE. While the physiological significance of these results remains to be established, they do suggest that CaBP‐D28k can activate enzymes and may be a regulator of yet to be identified target enzymes in certain tissues.

Studies on the amino and carboxyl terminal amino acid sequences of reovirus capsid polypeptides

Abstract Techniques are described for isolating several reovirus capsid polypeptides in amounts sufficient for determination of amino and carboxyl terminal amino acid sequences and fingerprinting. Among them are chromatography on CM- and DEAE-Sephadex in the presence of urea, and gel filtration on agarose A-15 m in the presence of sodium dodecyl sulfate. Using a combination of these procedures, and starting with either virions or cores, polypeptides σ3, σ2, and μ2 have been obtained essentially pure. Polypeptides λ1 and λ2 have been obtained as a mixture which has so far not been resolved. All reovirus capsid polypeptides except μ2 possess blocked amino terminal amino acid residues. The amino terminal amino acid sequence of polypeptide μ2 is H2N-Pro-Gly-Gly-Val-Pro-. This suggests that polypeptide μ2 is derived from its precursor, polypeptide μ1, by cleavage of the amino terminal portion of the polypeptide chain. The carboxyl terminal regions of at least three of the five major reovirus capsid polypeptides are different. Polypeptide σ3 ends in -(val, val, leu)-COOH; polypeptide μ2 in -leu-(arg, tyr, tyr)-Arg-COOH; and either one or both of the two polypeptides λ1 and λ2 terminate(s) in -Arg-COOH, the adjacent amino acid sequence being different from that of μ2.