Witold Drabikowski

Similarity in Ca2+-induced changes between troponin-C and protein activator of 3′:5′-cyclic nucleotide phosphodiesterase and their tryptic fragments

Abstract 1. 1.|The Ca2+-dependent protein activator of 3′:5′-cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) has been isolated from bovine brain and its properties have been compared with those of troponin-C. It has been shown that bovine brain does not contain troponin-C, but only the protein activator of phosphodiesterase. Its molecular weight is 16 500 as compared with 18 000 for troponin-C. 2. 2.|Similarly to troponin-C, calcium has a pronounced effect on the rate of cleavage of protein activator by trypsin and on the obtained peptide pattern. In the presence of 0.1 mM CaCl2 two large peptides are formed, essentially resistant to further splitting, whereas upon removal of calcium by chelators a fast cleavage of protein activator into small peptides takes place. 3. 3.|Similarly to troponin-C, tyrosine fluorescence intensity of protein activator is markedly enhanced in the presence of Ca2+. The apparent binding constant for Ca2+ calculated from the transition midpoint of fluorescence changes is about 1 · 107 M−1. During digestion of protein activator with trypsin in the presence of CaCl2 essentially no change of fluorescence intensity takes place and the subsequent decrease upon removal of calcium is reversible. 4. 4.|Two large peptides that accumulate during digestion by trypsin in the presence of Ca2+ of both troponin-C and protein activator have been isolated and their properties have been compared with those of corresponding parent molecules. One of the peptides obtained from both proteins retains the ability of the intact molecular to change the mobility on the polyacrylamide gel electrophoresis in the presence of urea depending on concentration of Ca2+ and to interacted with the inhibitory component of troponin in the presence of Ca2+. This peptide also shows Ca2+-dependent fluorescence changes, characteristic for parent molecule. 5. 5.|On the basis of similarity between the two proteins and their tryptic fragments it is suggested that in the presence of Ca2+ protein activator is cleaved in the same area as troponin-C. The peptide obtained from protein activator, which shows Ca2+-dependent changes characteristics for intact molecules, corresponds to the troponin-C peptide containing calcium binding sites 3 and 4. All these results furnish new pieces of evidence for a pronounced structural similarity between troponin-C and protein activator of cyclic nucleotide phosphodiesterase.

Filipin as a fluorescent probe for the location of cholesterol in the membranes of fragmented sarcoplasmic reticulum

Abstract A polyene antibiotic, filipin, when excited at 360 nm in solvents of low polarity, exhibits a strong fluorescence with a maximum at 480 nm. Mixed lecithin-cholesterol micelles, but not aqueous dispersions of cholesterol, also cause an increase of filipin fluorescence. Filipin binds to the vesicles of fragmented sarcoplasmic reticulum. The binding is considerably decreased after removal of cholesterol and abolished after lipid depletion. A restoration of filipin binding is achieved in the former case by rebinding of cholesterol, in the latter case only by the cholesterol-phospholipid micelles. Cholesterol additionally bound to intact vesicles does not cause an increase of binding of filipin. An enhancement of filipin fluorescence intensity is observed on its binding to the fragmented sarcoplasmic reticulum, indicating binding to regions of low polarity. Fluorescence with a maximum at about 340 nm, exhibited by sarcoplasmic reticulum vesicles by excitation at 280 nm, decreases in the presence of filipin with a concomitant appearence of fluorescence with a maximum at 480 nm. These results indicate an energy transfer from tryptophan residues to filipin.

Comparative studies on thermostability of calmodulin, skeletal muscle troponin C and their tryptic fragments

Received 7 January 1983 Abstract and keywords not received 1. INTRODUCTION Troponin C and calmodulin belong to the family of homologous intracellular calcium binding pro- teins. Each is composed of 4 calcium-binding do- mains. In the case of skeletal muscle troponin C, 2 of calcium-binding sites are high-affinity sites which also bind Mg2+ and 2 others are low- affinity, so-called ‘Ca’+-specific’ sites

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Endogenous proteinases in vertebrate skeletal muscle

1. 1. Activity of endogenous proteases in the muscles of various vertebrates was measured over a wide pH range. Three distinct regions of autolysis was found at acid, neutral and alkaline pH range. The activity in the latter pH was observed only in rat. mouse, and to a smaller extent in dog and human skeletal muscles. 2. 2. In the rat muscle the amount and size of peptides formed during autolysis as well as distribution of autolytic activity in the subcellular fractions was determined. 3. 3. Proteases working at acid pH range and localized in lyzosomes seem to degrade chiefly soluble sarcoplasmic and not myofibrillar proteins. 4. 4. Autolytic activity at alkaline pH is present in 0–600 g fractions. This protease degrades all myofibrillar proteins. Its activity is inhibited almost completely by diisopropyl-fluorophosphate and soy-bean trypsin inhibitor, partially by l-I-tosylamidophenylethylchloromethylketone and by injection of rats with the preparation “48/80”. All the results indicate that the alkaline protease is a chymotrypsin-like protease of mast-cell origin. 5. 5. The results suggest that at neutral pH range another proteolytic system is active in muscle. although its activity is rather low.

Localization of hydrophobic sites in calmodulin and skeletal muscle troponin C studied using tryptic fragments a simple method of their preparation

The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described.

Proton magnetic resonance studies on proteolytic fragments of troponin-C. Structural homology with the native molecule

Comparison of proton magnetic resonance spectra of a tryptic and a thrombin fragment of troponin-C with that of the native protein has identified the domain of the molecule influenced by Ca2+ binding to the lower affinity regions I and II of troponin-C. The binding of Ca2+ to these sites results in a subtle alteration of the tertiary fold of the N-terminal half of troponin-C involving weakened contacts between several hydrophobic groups. The role and kinetics of the movements within the troponin-C molecule associated with binding at the regulatory sites are discussed.

The origin of 30,000 dalton protein in troponin preparations

Received 17 November 197 2 1. Introduction It is already well established that the Ca2+ sensi- tivity of actomyosin is rendered by the regulatory protein system, composed of troponin and tropo- myosin [ 1,2] . Troponin appeared to consist of sever- al components which can be separated by DEAE- Sephadex chromatography [3,4] . We have recently shown [4,5] that out of three main constituents of troponin called: TN-C (mol. wt. 18,000), TN-I (mol. wt. 24,000) and TN-B (mol. wt. 39,000) (the average molecular weights and the nomenclature were taken from [5] ) two latter proteins are very susceptible to the proteolytic digestion. Thus, the other constituent of troponin corresponding to the protein of molecu- lar weight 13,000- 14,000 daltons, was found to be the product of catheptic degradation of TN-I, formed during preparation of troponin, namely during incuba- tion at pH 4.6 [4, S] . In addition, troponin prepara- tions contained usually variable amounts of another protein of molecular weight of about 30,000 daltons [4, 6-81 . We have recently found particularly large amounts of this protein in troponin preparations from slow and cardiac muscles [9] concominantly with considerable decrease of TN-B. The observation suggested that 30,000 component might derive from TN-B. This assumption obtained experimental evi- dence in the present work.

Role of cholesterol in the Ca2+ uptake and ATPase activity of fragmented sarcoplasmic reticulum

Abstract With the use of continuous sucrose density gradient a highly purified preparation of vesicles of fragmented sarcoplasmic reticulum was obtained and its lipid content was determined. Short mild treatment of fresh vesicles with aqueous diethyl ether or aqueous heptane removes only a part of the cholesterol. Aqueous heptane treatment does not lead, contrary to that of aqueous diethyl ether, to the loss of the ability of vesicles to actively accumulate Ca 2+ . Exhaustive extraction of lyophylized vesicles with dry diethyl ether or dry heptane enables removal of all cholesterol and most of the other neutral lipids without any changes in the Ca 2+ uptake ability. Thus, the results show that the loss of specific properties of fragmented sarcoplasmic reticulum as a result of treatment with aqueous diethyl ether is due to the specific action of this solvent but not a result of the removal of some part of cholesterol from the membranes. Contrary to phospholipids, cholesterol and other neutral lipids seem to be bound “loosely” to the membranes of sarcoplasmic reticulum; can be easily extracted with apolar solvents in the absence of water and do not play any direct role in the specific function of this biomembrane system. Cholesterol can be bound to the sarcoplasmic reticulum vesicles in large amounts.

Studies on the exchange of G-actin-bound calcium with bivalent cations

Abstract 1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu 2+ , action remains active, i.e. , contains bound ATP and polymerizes in 0.1 M KCl. 2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin. 3. The rate of exchange is of the same order for bivalent cations studied, including calcium. 4. G-actin-bound Ca 2+ is fully replaced, besides free Ca 2+ , by free Mn 2+ and Cd 2+ . The replacement with Mg 2+ , Co 2+ , Ni 2+ and Zn 2+ is not complete, and there is practically no reaction with Ba 2+ and Sr 2+ . 5. Assuming the affinity constant of Ca 2+ as 1, the following affinity constants for other bivalent cations were obtained: Mn 2+ , 0.90; Cd 2+ , 1.07; Mg 2+ , 0.27; Zn 2+ , 0.22; Co 2+ , 0.18; Ni 2+ , 0.08. 6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca 2+ .