Biomedical subjects

B M Martin

Publications and source records attributed to B M Martin.

At least 73 records · Page 4Linked to original sources

PubMed1995

A biochemical and immunocytochemical study on the targeting of alglucerase in murine liver.

A current hypothesis is that functional glucocerebrosidase needs to be delivered to the lysosomes of tissue macrophages to guarantee successful enzyme therapy for Gaucher's disease. In this study, biochemical and immunohistochemical techniques were applied to identify in mice the localization of intravenously administered alglucerase (human modified placental glucocerebrosidase). Only in liver and spleen was a significant increase of glucocerebrosidase activity observed, with a maximum level at 15 minutes after enzyme infusion. The uptake of enzyme by liver was sufficiently high to allow more detailed studies on the (sub)cellular distribution of human alglucerase. The enzyme in liver is localized both in the endosomal-lysosomal system of the Kupffer cells and the endothelial cells lining the lumen of the sinusoids. Uptake by both of these types of cell is prevented by mannan. The results suggest that the cellular mechanisms responsible for improvement of Gaucher patients receiving alglucerase treatment is probably more complicated than previously recognized.

Animals↗

PubMed1994

Novel K(+)-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch.

Two novel toxins were purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, using an immunoassay based on antibodies raised against noxiustoxin (NTX), a known K(+)-channel-blocker-peptide. The primary structure of C. l. limpidus toxin 1 was obtained by Edman degradation and was shown to be composed of 38 amino acid residues, containing six half-cystines. The first 36 residues of C. l. limpidus toxin 2 were also determined. Both toxins are capable of displacing the binding of radio-labelled NTX to rat brain synaptosomes with high affinity (about 100 pM). These toxins are capable of inhibiting transient K(+)-currents (resembling IA-type currents), in cultured rat cerebellar granule cells. About 50% of the peak currents are reduced by application of a 1.5 microM solution of toxins 1 and 2 The K+ current reduction is partially reversible, under washing but not voltage-dependent. Comparison of the primary structure of C. l. limpidus toxin 1 with other known toxins shows 74% identity with margatoxin, 64% with NTX, 51% with kaliotoxin, 39% with iberiotoxin, 37% with charybdotoxin and Lq2, and 29% with leirutoxin 1. The only invariant amino acids in all these toxins are the six cysteines, a glycine in position 26 and two lysines at positions 28 and 33, respectively. The relevance of these differences in terms of possible structure-function relationships is discussed.

Amino Acid Sequence↗

PubMed1994

Cloning, genomic organization, and osmotic response of the aldose reductase gene.

Diverse organisms accumulate organic osmolytes to adapt to hyperosmotic stress. The molecular basis of eukaryotic gene osmoregulation remains obscure. Aldose reductase [AR; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21], which catalyzes the conversion of glucose to sorbitol (an organic osmolyte), is induced in renal medullary cells under hyperosmotic conditions. Elevated extracellular NaCl increases AR mRNA transcription in PAP-HT25 cells, a cell line derived from the rabbit renal papilla. We have cloned and characterized the rabbit AR gene to determine how it is regulated by hyperosmolality. The length of the gene, not including 5' or 3' flanking regions, is approximately 14.7 kilobases (kb) organized into 10 exons and 9 introns. The transcription start site is 36 base pairs upstream of the initiator methionine codon. A 5-kb fragment containing approximately 3.5 kb of 5' flanking region was isolated. The 3.5-kb sequence was examined for basal promoter activity and hyperosmotic response in luciferase reporter gene constructs. A 235-base-pair fragment (base pairs -208 to +27) was able to drive the downstream reporter gene in transfected PAP-HT25 cells under isoosmotic conditions (300 mosmol/kg of H2O). When this fragment plus the remaining upstream sequence (from approximately base pair -3429 to base pair +27) was used, cells in hyperosmotic medium (500 mosmol/kg of H2O) showed about 40-fold induction of luciferase expression compared with cells in isoosmotic medium. The upstream fragment (from approximately base pair -3429 to base pair -192) also conferred osmotic response to a heterologous promoter (B19). This finding evidences putative osmotic response element(s) (OREs) within a specific DNA fragment in a eukaryotic genome. Identification and characterization of OREs within this fragment and their associated trans-acting factors should reveal the molecular mechanisms of gene regulation in osmotic stress.

Aldehyde Reductase↗

PubMed1994

Cloning and analysis of cDNA encoding a major airway glycoprotein, human tracheobronchial mucin (MUC5).

Two unique nucleotide probes for human tracheobronchial mucin glycoprotein (TBM) were generated via polymerase chain reaction with degenerate primers deduced from the TBM:TR-3A tryptic peptide sequence and were used to isolate a 3.6 kilobase cDNA, clone NP3a, from a human nasal polyp cDNA library. Clone NP3a was localized to chromosome 11 and contained a 3168 nucleotide open reading frame which encoded three TBM peptide fragments, thus confirming that clone NP3a partially encodes TBM. TBM also contains five tandem repeats of TTVGP/S and an octapeptide GQCGTCTN, which is conserved in human intestinal mucin MUC2 and rat intestinal mucin-like protein (MLP) suggesting that this sequence has a functional significance for secreted mucins. TBM has amino acid similarity to the cysteine-rich domains at the carboxyl termini of MUC2, rat MLP, bovine and porcine submaxillary mucins, and human von Willebrand factor. Strikingly, a large percentage of the cysteine residues in the overlaps are highly conserved: 90% in MUC2 and von Willebrand factor, 80% of bovine submaxillary mucin, 70% in porcine submaxillary mucin, and 64% in rat MLP, suggesting that conserved cysteines may be important for the tertiary structure of secreted glycoproteins. These studies demonstrate that clone NP3a is a candidate for MUC5, making it the only human mucin gene reported to date whose gene product has been isolated from airway secretions.

Amino Acid Sequence↗

PubMed1994

Substrate binding domains in pyruvate phosphate dikinase.

Proteolysis of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) in its free or phosphorylated state with subtilisin Carlsberg followed two different cleavage pathways. The major pathway involved initial cleavage of the holoenzyme (93 kDa) into a stable 25-kDa N-terminal fragment and transiently stable 67-kDa C-terminal fragment. The 67-kDa fragment was cleaved to generate a stable 35-kDa fragment and an unstable 30-kDa fragment (containing the catalytic histidine). Proteolytic cleavage via the minor pathway divided the holoenzyme into an unstable 37-kDa N-terminal piece (which was further cleaved to the stable 25-kDa fragment produced in the major pathway) and a transiently stable 55-kDa C-terminal fragment. The 55-kDa fragment was then cleaved to produce the stable 35-kDa fragment produced by the major pathway. The cleavage pattern of PPDK complexed with the ATP analog adenyl imidodiphosphate was identical to that of the free enzyme, only the rate of cleavage as slower. In contrast, proteolysis of the phosphorylenzyme-oxalate complex generated the 55-kDa fragment indicating that oxalate binding induces a change in protein conformation. Treatment of PPDK with [1-14C]bromopyruvate followed by proteolysis revealed selective radiolabeling of the stable 35-kDa fragment while similar experiments with [14C]2',3'-dialdehyde adenosine 5'-monophosphate resulted in selective radiolabeling of the stable 25-kDa fragment. These results were interpreted to suggest that PPDK contains several structural domains and that the catalytic histidine, the pyruvate binding site, and the ATP binding site may be located on different domains.

Adenosine Triphosphate↗

PubMed1994

Isolation and characterization of a novel toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch.

A novel peptide, toxic to mice, was purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, by means of gel filtration and ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The complete amino acid sequence was determined by automatic Edman degradation of reduced and alkylated toxin, and by overlapping sequences of fragments of the toxin, generated by cleavage with proteinase V8 separated by HPLC. This toxin is composed of 66 amino acid residues, contains eight half-cystine residues, and is highly similar (91%) to the amino acid sequence deduced for toxin 1 of C. limpidus tecomanus and toxin 4 from C. noxius venom (89%). This peptide displaces the binding of radiolabeled toxin 2 of C. noxius from synaptosomal membranes of rat brain with superimposable kinetics, supporting the conclusion that it belongs to the beta-scorpion toxin class. Further characterization of C. l. limpidus toxin 1, as we have named it, was performed by means of competition experiments with monoclonal antibodies and various purified scorpion toxins, using an ELISA assay. A panel of six distinct monoclonal antibodies (mAB) against toxin 2 and 3 of C. noxius was used. From these, only three clones, originally named BCF1, BCF8 and BCF9, were able to recognize toxin 1 from C. l. limpidus.

Amino Acid Sequence↗

PubMed1994

Isolation of a toxin from Centruroides infamatus infamatus Koch scorpion venom that modifies Na+ permeability on chick dorsal root ganglion cells.

A novel toxin was isolated and characterized from the venom of the Mexican scorpion Centruroides infamatus infamatus. It has an apparent mol. wt of 7600, compatible with the presence of 66 amino acid residues per molecule. The N-terminal amino acid sequence was determined (up to residue 48) and showed approximately 95% similarity with toxins from other Mexican scorpions of the gnus Centruroides. Experiments conducted with chick dorsal root ganglion cells showed that toxin 1 is a Na+ channel effector, causing a decrease in the peak Na+ permeability, similar to decreases observed for typical beta-scorpion toxins.

Amino Acid Sequence↗

PubMed1994

Characteristics of a purified dog hepatic microsomal N,O-acyltransferase.

Dog liver microsomes have at least three different enzymes that are capable of the deacylation of amides, N-arylhydroxamic acids and carboxylesters, the acyltransfer of N-arylhydroxamic acids and the N-acetylation of arylamines. As judged by SDS-PAGE stained with silver nitrate, one of these enzymes was purified to homogeneity by sequential treatment with Triton X-100, ion-exchange column chromatography, gel filtration and chromatofocusing. The protein was a glycoprotein trimer with a subunit weight of approximately 60 kDa. It showed microheterogeneity on analytical isoelectric focusing (IEF) in polyacrylamide with pls of 5.4-5.6. Following digestion with endoglycosidase H, its subunit weight was reduced to approximately 58 kDa, and it appeared to be homogeneous on IEF with a pl of approximately 5.6. A monoclonal antibody prepared against this enzyme also reacted with the pl 6.0 carboxylesterase of rat liver microsomes, but did not react with the other two dog hepatic acyltransferases. Conversely, a polyclonal antibody raised against the rat esterase reacted with the dog enzyme. The N-terminal sequence of the enzyme was Y-P-S-L-P-P-V-V-D-T-V-Q-G-K-V-, which was homologous to the form 1 carboxylesterase of rabbit liver and the pl 6.0 carboxylesterase of rat liver. Immunohistochemical analyses showed the presence of this enzyme in the epithelium of dog liver and urinary bladder, human liver and rat liver, esophagus, forestomach, glandular stomach, small and large intestines, renal tubules, trachea and prostate and alveolar cells of lung. Since this enzyme is present in the urothelium, it may be important for the activation of urinary metabolites of carcinogenic arylamines for the initiation of bladder carcinogenesis in the dog.

Acyltransferases↗

PubMed1994

Isolation and pharmacological characterization of four novel Na+ channel-blocking toxins from the scorpion Centruroides noxius Hoffmann.

Four novel Na+ channel-blocking toxins (numbered 6 to 9) were purified from the venom of the scorpion Centruroides noxius Hoffmann by gel filtration and high-performance liquid chromatography and their chemical and pharmacological properties were characterized. Amino acid analysis and SDS-PAGE of the pure toxins showed them to be composed of approximately 65 amino acid residues with a molecular mass of approximately 7,500 Da. The amino acid sequences of the newly isolated toxins displayed substantial similarity to those of previously isolated and characterized C. noxius toxins. Toxin 7, the most active toxin from this venom, selectively blocked the whole-cell inward Na+ current (INa) from guinea pig ventricular myocytes without altering the K+ or Ca2+ currents. Using rat brain synaptosomes, the specific binding parameters of 125I-Toxin 7 were determined: KD = 40 pM and Bmax = 1.8 pmol/mg protein. The binding was independent of membrane potential and was displaced by all Na+ channel-toxins from C. noxius venom, with K0.5s ranging from 60 pM to 60 nM. Tityus gamma-toxin, a toxin representative of beta-scorpion toxins, totally displaced 125I-Toxin 7 binding, but AaH II and Lqq V toxins, representative of alpha-scorpion toxins, had no effect. All four C. noxius toxins inhibited [3H]GABA uptake by synaptosomes with IC50 s similar to KD s. The toxin effect was not synergistic with veratridine. From these results, it was concluded that the newly purified toxins exert their effects by binding to Site 4 of the voltage-sensitive Na+ channel and must, therefore, be classified as beta-scorpion toxins.

Amino Acid Sequence↗

PubMed1994

Characteristics of a Brucella species from a bottlenose dolphin (Tursiops truncatus).

A culture isolated from an aborted fetus of a bottlenose dolphin (Tursiops truncatus) was characterized. The isolate was a gram-negative coccobacillus, and the colonial morphology was typical of a smooth Brucella. The isolate was positive for catalase, oxidase, nitrate reduction, and urease. Hydrogen sulfide was not produced. It grew in air at 37 C but required 72 hours for good growth. There was growth on media containing basic fuchsin, thionin, thionin blue, penicillin, and erythritol. The M antigen was dominant, and the isolate was lysed by 4 of 10 brucellaphages tested. The oxidative metabolic profile of the isolate was similar to that for B. abortus but differed in utilization of L-asparagine, L-glutamic acid, and DL-citrulline. Whole-cell lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles were markedly different from the protein profiles of reference strains of Brucella species. Biochemical and oxidative metabolism profiles indicated that the isolate belongs in the genus Brucella but did not match the profiles of any established species or biovars. This isolate may be an atypical strain of a recognized Brucella species or a new biovar or species of Brucella.

Animals↗

PubMed1993

The genomic region encoding toxin gamma from the scorpion Tityus serrulatus contains an intron.

The gene encoding toxin gamma from the scorpion, Tityus serrulatus, was amplified by PCR from genomic DNA employing synthetic oligonucleotides designed from the reported cDNA sequence. The nucleotide sequence of this gene reveals the presence of an intron of 475 base pairs (bp) which interrupts the region that encodes the signal peptide of the precursor toxin. A comparison of the intron boundary sequences of the gamma toxin gene with ones from other arachnid genes is also presented.

Animals↗

PubMed1993

Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes.

The major heat-stable shrimp allergen (designated as Sa-II), capable of provoking IgE-mediated immediate type hypersensitivity reactions after the ingestion of cooked shrimp, has been shown to be a 34-kDa heat-stable protein containing 300 amino acid residues. Here, we report that a comparison of amino acid sequences of different peptides generated by proteolysis of Sa-II revealed an 86% homology with tropomyosin from Drosophila melanogaster, suggesting that Sa-II could be the shrimp muscle protein tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from uncooked shrimp (Penaeus indicus) and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8 to 5.4. In the presence of 6 M urea, the mobility of both Sa-II and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp-sensitive patients as assessed by competitive ELISA inhibition and Western blot analysis. Tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were superimposable. Dot-blot and competitive ELISA inhibition using sera of shrimp-sensitive patients revealed that antigenic as well as allergenic activities were associated with two peptide fractions. These IgE-binding tryptic peptides were purified and sequenced. Mouse anti-anti-idiotypic antibodies raised against Sa-II specific human idiotypic antibodies recognized not only tropomyosin but also the two allergenic peptides, thus suggesting that these peptides represent the major IgE binding epitopes of tropomyosin. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes (residues 50-66 and 153-161) with the corresponding regions of tropomyosins from different vertebrates confirmed lack of allergenic cross-reactivity between tropomyosins from phylogenetically distinct species.

Allergens↗

PubMed1993

Isolation of two distinct 3-hydroxysteroid sulfotransferases from the guinea pig adrenal. Evidence for 3 alpha-hydroxy versus 3 beta-hydroxy stereospecificity.

Two physically distinct hydroxysteroid sulfotransferases (HSTs) that demonstrate substrate specificity with respect to the orientation of the ring A 3-hydroxyl group have been isolated from the guinea pig adrenal gland. Nondenaturing liquid-phase isoelectric focusing permitted the separation of the activities, and reverse phase high performance liquid chromatography was used to purify the two proteins to homogeneity. The 3 beta-HST had an apparent molecular mass of 33 kDa and utilized pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone as substrates. The 3 alpha-HST was slightly smaller at 32 kDa and utilized allopregnanolone and androsterone as substrates. The proteins were further distinguished by isoelectric point, immunoreactivity, and tryptic peptide mapping. Peptides isolated from both guinea pig HSTs demonstrated significant amino acid sequence homology (approximately 65% identity) to rat liver HST; however, available sequence data from the two proteins did not yield differences that might account for their stereospecific substrate selectivity. This paper represents the first definitive report demonstrating the existence of discrete HSTs that exhibit substrate specificity based on the stereochemistry of the 3-hydroxyl group.

Adrenal Glands↗

PubMed1993

Phosphorylation of human and bovine prothymosin alpha in vivo.

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.

Alkaline Phosphatase↗

PubMed1993

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Amino Acid Sequence↗

PubMed1993

Primary structure determination and cloning of the cDNA encoding toxin 4 of the scorpion Centruroides noxius Hoffmann.

A peptide (toxin II-10), shown to be a Na+ channel blocker, was purified from the venom of the scorpion Centruroides noxius Hoffmann and sequenced by Edman degradation. It has 66 amino acid residues with the C-terminal residue (asparagine) amidated, as demonstrated by mass spectrometry. In addition, we report the cloning and the nucleotide sequence of the cDNA (CngtV) that codes for this toxin. We discuss the mechanism for processing the precursor peptide to its final form and compare the primary structure to that of other Na+ channel toxins. Two distinct groups of toxins seem to emerge from this comparison, suggesting a structure-function relationship of these peptides towards the recognition of either mammalian or insect tissues.

Amino Acid Sequence↗

PubMed1993

Effect of saposins on acid sphingomyelinase.

The effect of saposins (A, B, C and D) on acid sphingomyelinase activity was determined using a crude human kidney sphingomyelinase preparation and a purified sphingomyelinase preparation from human placenta. Saposin D stimulated the activity of the crude enzyme by increasing its apparent Km and Vmax. values for sphingomyelin hydrolysis. Unlike the crude enzyme, the activity of the purified enzyme was strongly inhibited by saposin D as well as other saposins. Saposin D decreased the apparent Km and Vmax values of purified sphingomyelinase activity. The effects of saposin D on the activity of different sphingomyelinase preparations appear to depend on Triton X-100, which is present in the crude enzyme but not in the purified enzyme. When the detergent was removed from the crude preparation, the effect of saposin D changed from being stimulatory to inhibitory. Conversely, when the detergent is added to the purified enzyme, the effect of saposin D on sphingomyelinase activity changed from being inhibitory to stimulatory. While other saposins were inhibitory or had no effect on sphingomyelinase activity in the above assay system, not only saposin D but also saposins A and C exhibited a stimulatory effect upon purified sphingomyelinase activity when the substrate, sphingomyelin, was added in the form of liposomes without detergent. Saposin B was not only inhibitory in the liposome system, but also reduced the stimulatory effect of saposins A, C and D. These observations indicate that the stimulatory effect of saposins A, C and D on acid sphingomyelinase activity is greatly influenced by the physical environment of the enzyme and suggest that similar effects by saposins may be exerted in lysosomal membranes.

Detergents↗

PubMed1993

Regulation of actomyosin interactions in Limulus muscle proteins.

Contraction of striated muscle from Limulus polyphemus, the horseshoe crab, is regulated by both calcium binding to a troponin-tropomyosin-dependent thin filament array and a myosin light chain kinase-dependent phosphorylation of myosin. We have isolated myosin from Limulus striated muscle and examined how these two regulatory systems affect the sliding velocity of actin filaments over myosin, using an in vitro motility assay. Our results show that in the presence of ATP, Limulus myosin must be phosphorylated in order to move actin filaments. No movement was observed for actin filaments interacting with dephosphorylated Limulus myosin. Calcium was not required for actin movement. In contrast, when both troponin and tropomyosin are bound to actin filaments, calcium is required for the movement of actin filaments over phosphorylated myosin. These results demonstrate that the "off" state of either the thin filament or thick filament regulatory system is dominant and that for the movement to occur, both phosphorylated Limulus myosin and an activated troponin-tropomyosin system are required. Tropomyosin by itself increases the sliding velocity of actin filaments over phosphorylated Limulus myosin about 10-fold in a calcium-independent manner. Tropomyosins from turkey gizzard smooth muscle, bovine cardiac muscle, and Limulus muscle all have a profound effect in increasing the velocity. Troponin alone does not change the velocity. Partial sequences of the tryptic phosphopeptides of Limulus myosin regulatory light chains generated following the phosphorylation by gizzard myosin light chain kinase yield ATS(PO4)NVFAMFEQNQIA for 21 kDa and SGS(PO4)NVFSMFT for 31-kDa light chain.

Actins↗