Evolutionary diversification of structure and function in the family of intracellular calcium-binding proteins

Summary The maximum parsimony method was used to reconstruct the genealogical history of the family of intracellular calcium-binding proteins represented by six major present-day lineages, three of which - calcium dependent modulator protein, heart and skeletal muscle troponin Cs, and alkali light chains of myosin - were found to share a closer kinship with one another than with the other lineages. Similarly, parvalbumins and regulatory light chains of myosin were depicted as more closely related, whereas the branch of intestinal calcium-binding protein proved to have the most distant separation. The computer-generated amino acid sequence for the common ancestor of these six lineages described a four domain protein in which each domain of approximately 40 amino acid residues had a mid-region, 12 residue segment that bound calcium and had properties most resembling those of the calcium dependent modulator protein. It could then be deduced that parvalbumins evolved by deletion of domain I, inactivation of calcium-binding properties in domain II, and acquisition of increased affinity for Ca⁺⁺ and Mg⁺⁺ in domains III and IV. Regulatory light chains of myosin lost the cation binding property from three domains, retaining it in I, whereas alkali light chains of myosin lost this ability from each of the four domains. In skeletal muscle troponin C all domains retained their calcium-binding activity; however, like parvalbumins, domains III and IV acquired high affinity properties. Cardiac troponin C lost its binding activity from domain I but otherwise resembled the skeletal muscle form. Finally, intestinal calcium-binding protein evolved by deletion of domains III and IV.Positive selection could be implicated in these evolutionary changes in that the rate of fixation of mutations substantially increased in the mid portions of those domains which were loosing calcium-binding activity. Likewise, when the cation binding sites were changing from low to high affinity, an accelerated rate of fixed mutations was observed. Once this new functional parameter was selected these regions showed a remarkable conservatism, as did those binding sites which were maintaining the lower affinity. Moreover even in sequence regions not directly involved in cation binding, the lineage of troponin C became very conservative over the past 300 million years, perhaps because of the necessity for maintaining specific interfaces in order for the molecule to interact with troponin I and T in a functional thin myofilament. A similar phenomenon was observed in domain II of the regulatory light chains of the myosin lineage suggesting a possible binding site with the heavy chain of myosin.This paper is dedicated to the memory of Jean-Francois Pechère, deceased     

The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins

We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C.     

小清蛋白研究进展

小清蛋白作为维持细胞内钙离子交换的钙结合蛋白,是脊椎动物体内肌浆蛋白的主要组成部分,在调节细胞内钙离子交换中起重要作用。我们简要综述了小清蛋白的分布、分类、结构和作用机理等,重点介绍小清蛋白在鱼肌肉运动、系统发育和神经组织中的作用以及由小清蛋白引起的过敏反应。     

Electrophoretic study of muscle extracts of low ionic strength]

The method of Na-dodecylsulfate electrophoresis in polyacrylamide gel demonstrated that the so-called tonoactomyosin of smooth muscles extracted from the muscle homogenate with salt media of low ionic power represented a complicated protein system into whose composition there was included a heavy myosin chain with the mol wt of 210000, premyosin subunit with the mol wt of 230000, actin, and, possibly, a number of other proteins. The extracts of low ionic power possessed Mg2+ and Ca2+ activated by ATP-ase activity. The premyosin subunit was also revealed in the extracts of low ionic power from the skeletal muscle homogenates. It is supposed that premyosin subunit was included into the enzymatic system responsible for the ATP-asic properties of the extracts of low ionic power from the homogenates of different types of muscles.     

Structural and functional properties of the non-muscle tropomyosins

Summary The non-muscle tropomyosins (TMs), isolated from such tissues as platelets, brain and thyroid, are structurally very similar to the muscle TMs, being composed of two highly -helical subunits wound around each other to form a rod-like molecule. The non-muscle TMs are shorter than the muscle TMs; sequence analysis demonstrates that each subunit of equine platelet TM consists of 247 amino acids, 37 fewer than for skeletal muscle TM. The major differences in sequence between platelet and skeletal muscle TM are found near the amino and carboxyl terminal ends of the proteins. Probably as the result of such alterations, the non-muscle TMs aggregate in a linear end-to-end manner much more weakly than do the muscle TMs. Since end-to-end interactions are responsible for the highly cooperative manner in which TM binds to actin, the non-muscle TMs have a lower affinity for actin filaments than do the muscle TMs. However, the attachment of other proteins to actin (e.g. the Tn-I subunit of skeletal muscle troponin or the S-1 subfragment of skeletal muscle myosin) can increase the affinity of actin filaments for non-muscle TM. The non-muscle TMs interact functionally with the Tn-I component of skeletal muscle troponin to inhibit the ATPase activity of muscle actomyosin and with whole troponin to regulate the muscle actomyosin ATPase in a Ca⁺⁺-dependent manner, even though one of the binding sites for troponin on skeletal TM is missing in non-muscle TM. A novel actomyosin regulatory system can be produced using Tn-I, calmodulin and non-muscle TM; in this case inhibition is released when the non-muscle TM detaches from the actin filament in the presence of Ca⁺⁺. Although it has not yet been demonstrated that the non-muscle TMs participate in a Ca⁺⁺-dependent contractile regulatory system in vivo it does appear that they are associated with actin filaments in vivo.     

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases.

1. Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.     

Troponin from nematode: purification and characterization of troponin from Ascaris body wall muscle

• 1.1. A troponin-like protein was isolated from body wall muscle of Ascaris and separated into three components, the mol. wts of which were approx. 58,000, 36,000 and 20,000 respectively.
• 2.2. The three components were designated as troponin-T (TNT), troponin-I (TNI) and troponin-C (TNC) in order of mol. wt, since each component had properties similar to the respective components of vertebrate skeletal-muscle troponin.
• 3.3. Ascaris troponin were localized on actin filaments with a 44 nm repeat, an approximately 4 nm longer repeat than vertebrate troponin.

     

A calorimetric study on calcium binding by troponin C from bullfrog skeletal muscle

Microcalorimetric titrations of bullfrog (Rana catesbeiana) skeletal troponin C with Ca2+ were carried out in the absence of Mg2+ at 25 degrees C and at pH 7.0. The observed enthalpy titration curve was divided into three stages. The first stage of the titration (up to 2 mol of Ca2+/mol of protein) was characterized as an extremely exothermic process (delta H = -52 kJ/mol of site), the second one (titration from 2 to 3 mol of Ca2+/mol of protein) as a weakly endothermic process (delta H = +26 kJ/mol of site), and the final one (over 3 mol of Ca2+/mol of protein) as a moderately exothermic process (delta H = -35 kJ/mol of site). The endothermic process of Ca2+ binding to the third site (the second stage) has the same property as that of the Ca2+ binding to every site of calmodulin but is distinctly different from those of the calmodulin-trifluoperazine complex and parvalbumins. This may suggest that an endothermic nature of Ca2+ binding, the reaction being driven solely by entropy change, is characteristic of the regulatory reactions of Ca2+ binding proteins accompanying the interaction with other proteins. The third Ca2+ binding site of bullfrog troponin C is, therefore, possibly involved in the regulation of muscle contraction.     

Identification and characterization of new protein chemoattractants in the frog skin secretome

The vomeronasal organ is a chemosensory organ present in most vertebrates and involved in chemical communication. In the last decade, the deciphering of the signal transduction process of this organ has progressed. However, less is known about the vomeronasal organ ligands and their structure-function relationships. Snakes possess a highly developed vomeronasal system that is used in various behaviors such as mating, predator detection, or prey selection, making this group a suitable model for study of the vomeronasal chemoreception. In this work, we used a proteomics approach to identify and characterize proteins from frog cutaneous mucus proteome involved in prey recognition by snakes of the genus Thamnophis. Herein we report the purification and characterization of two proteins isolated from the frog skin secretome that elicit the vomeronasal organ-mediated predatory behavior of Thamnophis marcianus. These proteins are members of the parvalbumin family, which are calcium-binding proteins generally associated to muscular and nervous tissues. This is the first report that demonstrates parvalbumins are not strictly restricted to intracellular compartments and can also be isolated from exocrine secretions. Purified parvalbumins from frog muscle and mucus revealed identical chemoattractive properties for T. marcianus. Snake bioassay revealed the Ca(2+)/Mg(2+) dependence of the bioactivity of parvalbumins. So parvalbumins appear to be new candidate ligands of the vomeronasal organ.     

Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.     

Calcium-binding proteins in the vorticellid spasmoneme

The proteins of the contractile spasmoneme from Vorticella convallaria, Carcheslium polypinum, and Zoothamnium geniculatum have been extracted in the detergent, sodium dodecyl sulfate (SDS), as well as urea and guanidine hydrochloride (GuCl). After SDS extraction, the molecular weight distribution of the proteins was examined by means of SDS-polyacrylamide gel electrophoresis. Significant amounts of material corresponding to the contractile proteins actin and tubulin are not present. The contractile organelles in the three species examined contain a group of closely related proteins of molecular weight near 20,000, which constitute a major part (40-60%) of the dry mass. The 20,000 mol wt proteins in Zoothamnium bind calcium with high affinity (pK congruent to 6) and are termed "spasmins." By means of urea polyacrylamide gel electrophorsis, it is demonstrated that in Carchesium and Zoothamnium certain spasmin components bind calcium even in the presence of 6 M urea. The binding of calcium in 6 M urea suggests a functional relationship between the spasmins and the calcium-binding proteins of striated muscle which behave similarly. The calcium binding in urea also indicates that the spasmins within a single spasmoneme have different calcium affinities, and this difference in calcium-binding properties may be an important factor in the physiological function of the organelle.     

Troponin Is Unlikely to Occur in Bovine and Chick Forebrain

A search for the presence of troponin in brain reveals that troponin is below 0.00037% of total bovine brain soluble protein. Troponin levels were examined using G-actin-linked Sepharose affinity chromatography and 45Ca binding. The chromatographic and 45Ca binding experiments revealed the presence of several actin and calcium-binding proteins, none of which corresponded to any troponin subunit. In addition, troponin was not found in any chick brain subfraction analyzed, and the level of troponin in chick nerve ending cytoplasm enriched for troponin was less than 0.023%. Considering that substantial amounts of myosin and actin occur in brain, these findings indicate that troponin is not likely to be a regulator of putative brain actomyosin interactions. The significance of these results and their relation to proposed models for neurotransmitter release is discussed.     

Effects of storage on activity and subunit structure of rabbit skeletal-muscle AMP deaminase.

On storage, AMP deaminase is converted into a form exhibiting hyperbolic kinetics even at low KCl concentration. This effect results from cleavage of the enzyme subunit (mol.wt. 79 000) to a product of similar size to the component of approx. mol.wt. 70 000 present in trace amounts in AMP deaminase just prepared from fresh muscle.     

Contractile Proteins of a Benthic Fish. III. Subunit Composition of Myosin

SYNOPSIS. Ultracentrifugal and electrophoretic experiments arereported on the subunit composition of myosin from skeletalmuscle of a benthic fish, Coryphaenoides species. Coryphaenoidesmyosin undergoes extensive association in concentrated KGI solutionsat neutral pH, but sedimentation equilibrium experiments indicatethe presence of a small fraction (3%) of monomeric myosin withmolecular weight approximately 440,000. At pH 11, some of theaggregated myosin is dissociated, and monomeric myosin is itselfdissociated into a heavy component (410,000 mol wt) and a lightcomponent (14,000 mol wt) that comprises 5–7% of the protein.The lialkali component of Coryphaenoides myosin yields a singlepredominant band on cellulose acetate electrophoresis and SDS-ureaelectrophoresis in 9% acrylamide gel. The stoichiometric evidenceindicates that Coryphaenoides myosin contains two heavy chains(205,000 mol wt) and two light chains (14,000 mol wt) that areequivalent with respect to net electrostatic charge and molecularweight. Preparations of myosin obtained by direct extractionfrom muscle mince and by dissociation of actomyosin extractedfrom muscle mince also contain 5% of a 47,000 mol wt componentpresumably actin), traces of 34–36,000 mol wt component,and about 5.7% of low molecular weight material (10,000–15,000)that probably represents contaminant protein, although the possibilityof denatured nivosin subunits cannot be excluded.     

Structural similarities between the Ca2+-dependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomyosin ATPase.

Results of studies of the Ca2+-dependent protein modulator of 3':5'-cyclic nucleotide phosphodiesterase isolated from bovine brain are presented which show its structural similarity to the Ca2+-binding subunit of muscle troponin. Both proteins have blocked NH2 termini, similar and characteristic ultraviolet absorption spectra, similar Ca2+-binding properties, very similar amino acid compositions, and co-migrate on sodium dodecyl sulfate-polyacrylamide gels. The primary structures of selected tryptic peptides isolated from bovine brain modulator protein are similar or identical with regions of the primary sequences of rabbit skeletal muscle and bovine cardiac muscle troponin C. Bovine brain modulator protein contains and unidentified ninhydrin-positive basic compound not found in muscle troponin C. An improved procedure is presented which yields 40 to 70 mg of modulator protein per kg of bovine brain.     

Conformational studies on parvalbumins by circular dichroism.

Structural variations of two parvalbumins, Whiting III and Pike III, in various denaturing conditions, have been studied by circular dichroism. CD signals are depressed from 4 urea. For Pike III, acidic pH, sodium dodecyl sulfate or complete removal of Ca2+ show little effect in the far ultraviolet region but rather strong effects in the near ultraviolet. For Whiting III similar results are obtained at acidic pH. Carboxymethylated Whiting III (0.15 Ca2+/mol) shows, on the contrary, decreased CD signals in the far and in the near ultraviolet spectra. Addition of Ca2+ fully restores the native CD spectra in both proteins. Ca2+ binding produces structural modifications which are found to vary according to parvalbumin and which seem in any case different from those described for troponin C.     

The contractile and regulatory proteins of insect flight muscle

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.     

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.     

Modulation of contractile activation in skeletal muscle by a calcium-insensitive troponin C mutant

Calcium controls the level of muscle activation via interactions with the troponin complex. Replacement of the native, skeletal calcium-binding subunit of troponin, troponin C, with mixtures of functional cardiac and mutant cardiac troponin C insensitive to calcium and permanently inactive provides a novel method to alter the number of myosin cross-bridges capable of binding to the actin filament. Extraction of skeletal troponin C and replacement with functional and mutant cardiac troponin C were used to evaluate the relationship between the extent of thin filament activation (fractional calcium binding), isometric force, and the rate of force generation in muscle fibers independent of the calcium concentration. The experiments showed a direct, linear relationship between force and the number of cross-bridges attaching to the thin filament. Further, above 35% maximal isometric activation, following partial replacement with mixtures of cardiac and mutant troponin C, the rate of force generation was independent of the number of actin sites available for cross-bridge interaction at saturating calcium concentrations. This contrasts with the marked decrease in the rate of force generation when force was reduced by decreasing the calcium concentration. The results are consistent with hypotheses proposing that calcium controls the transition between weakly and strongly bound cross-bridge states.     

Erythrocyte troponin inhibitor-like protein: Isolation and characterization

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000–24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca²⁺ and K⁺-EDTA ATPase activity. Furthermore, they inhibited actin-activated Mg²⁺-ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium-binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor-like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor-like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.