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At least 73 records · Page 4Linked to original sources
PubMed1987

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain.

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.

Amino Acid Sequence↗
PubMed1986

Kinetic and equilibrium studies of the interaction of amsacrine and anilino ring-substituted analogues with DNA.

The kinetic stability of complexes of the clinical antileukemic drug amsacrine and anilino ring-substituted analogues with DNA has been studied by using the surfactant sequestration technique in a stopped-flow spectrophotometer. In addition, viscometric measurements using covalently closed circular DNA and displacement measurements of DNA-bound ethidium have been performed to evaluate helix unwinding angles and association constants, respectively. Amsacrine and its analogues dissociate from DNA by a complex kinetic pathway which involves at least three discernible transiently bound forms of the drug. Dissociation time constants for amsacrine are found to range from less than 1 to 6 ms in buffer of ionic strength 0.1, and the biomolecular association rate constant is greater than 10(6) M-1 s-1. We find that amsacrine forms one of the weakest intercalation complexes among the compounds studied, as judged by the criteria of kinetic stability, affinity, and chromophore-base pair stacking interactions. Unlike other compounds of this broad class (intercalating chromophores bearing sterically-demanding side chains), addition of the bulky side chain has little effect on the kinetic stability of the drug-DNA complexes, suggesting that the acridinesulfonanilides may intercalate into DNA from the less sterically demanding major groove of the helix. Such a binding geometry would facilitate the formation of the previously proposed ternary complex between amsacrine, DNA, and gene-regulatory enzymes (e.g., topoisomerases and repressors) which are known to bind in the major groove.

Aminoacridines↗
PubMed1998

Tapping mode atomic force microscopy of hyaluronan: extended and intramolecularly interacting chains.

The extracellular matrix polysaccharide hyaluronan has been examined by tapping mode atomic force microscopy. High molecular weight hyaluronan was deposited on mica from dilute aqueous solution and imaged in air. Long unbranched chains could be observed and were found to be compatible with the known covalent structure of hyaluronan. In addition, chains with evidence of intramolecular association were observed. In the simplest cases, the association took the form of loops stabilized by antiparallel double-stranded (probably double-helical) segments. In other cases, the polarity of the associated regions could not be determined. Extensive intramolecular association in long hyaluronan chains resulted in a fenestrated structure of the same type as that formed by intermolecular association at higher concentrations.

Animals↗
PubMed1988

Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV.

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.

Amino Acid Sequence↗
PubMed1981

Human plasma kallikrein. A rapid purification method with high yield.

A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).

Chromatography, Affinity↗
PubMed1980

[Hemoglobins, XXXII. Analysis of the primary structure of the monomeric hemoglobin CTT VIIA (erythrocruorin) or Chironomus thummi thummi, Diptera (author's transl)].

The dimeric hemoglobin CTT VIIA (erythrocruorin) was isolated from the hemolymph of the larva from Chironomus thummi thummi and purified by preparative polyacrylamide gel electrophoresis. Peptides obtained by limited tryptical digestion were sequenced by automatic Edman degradation. For the elucidation of the sequence in the C-terminal region of the chain, additional cleavages with proteinase of Staphylococcus aureus and chymotrypsin were necessary. CTT VIIA is compared with human beta-chains and other hemoglobins of Chironomus. The amino acid residues in the pocket are especially discussed. Most of them are invariant in all Chironomus hemoglobins, independent of the size of the heme pocket, which is normal in some components and enlarged in others.

Amino Acid Sequence↗
PubMed1986

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions.

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.

Animals↗
PubMed1990

Differential expression of laminin A and B chains during development of embryonic mouse organs.

Laminin is a large glycoprotein of basement membranes. The best described laminin from a mouse tumor contains three polypeptide chains (A, B1 and B2), but there is recent evidence that some cell types produce laminin isoforms lacking the A chain. We have here studied the occurrence of the isoforms during mouse organogenesis. In all tissues studied, the A chain mRNA and polypeptide were more weakly expressed than those of the B chains. Laminin A chain polypeptides showed a much more restricted tissue distribution than the B chains. Laminin A chain polypeptide was mainly detected in basement membranes of epithelial cells, suggesting that this chain is important for morphogenesis of epithelial sheets. Most endothelial basement membranes and all embryonic mesenchyme matrices studied seemed to lack the A chain even though they contained B chains. Several of the cells producing laminin devoid of A chain seem to produce other polypeptides that become complexed to the B chains. With an anti-laminin antiserum, which in immunoblots reacts only with A and B polypeptide chains, additional polypeptides of 160 and 190 x 10(3) Mr were co-precipitated from all tissues studied. In developing heart, a polypeptide of 300 x 10(3) Mr was co-precipitated in addition. Our data suggest that these laminin-associated polypeptides are not formed by a differential splicing of the known A chain mRNA. Northern blotting of poly (A)+ RNA showed only 10kb A chain transcripts but no truncated forms. We conclude that several cell types in the mouse embryo produce laminin variants that lack the 400 x 10(3) Mr A chain. Since a major cell binding site of laminin contains parts of the A chain, the variants should differ in biological function from laminin containing this A chain.

Animals↗
PubMed1989

Human placental Fc gamma receptor. Studies on affinity labeling of the receptor with IgG.

The aim of the presented work was to find out whether the Fc gamma receptor from human placental syncytiotrophoblast plasma membranes in its native membrane-bound state is composed of more subunit chains than previously found in our laboratory in the purified receptor. The chains might be lost during the purification procedure with the use of various chaotropic reagents, e.g. acidic or alkaline buffers, detergents. To study this problem, affinity labeling technique and bifunctional crosslinking reagents were used to covalently link IgG with the Fc gamma receptor. The reagents used were: noncleavable dimethylsuberimidate (DMS), cleavable dimethyl 3,3-dithiobispropionimidate (DTBP), and photoactivable sulfosuccinimidyl 6-/4' azido-2'nitrophenylamino/hexanoate (sulfo-SANPAH). Human 125I-IgG were crosslinked to the membrane-bound receptor or unlabeled IgG was crosslinked to 3H-labeled placental membranes. When 125I-IgG were used in the presence of an excess of unlabeled IgG, recovery of radioactivity after crosslinking was much lower, indicating that human IgG and the placental Fc gamma receptor were involved in the formation of crosslinking of ligand-receptor complexes. The products of crosslinking were analyzed in SDS-PAGE. In the absence of reducing agents, high molecular weight products were present which did not enter the gel or formed broad diffused bands at the top of the gel. Therefore, the products of crosslinking were analyzed under reducing conditions. Analysis by SDS-PAGE demonstrated a major polypeptide band Mr of 160,000 in soluble products of crosslinking of IgG to the placental Fc gamma receptor, regardless of which noncleavable crosslinker was used. The protein is built either of two molecules of the receptor subunit chain, Mr of 60,000 and one IgG heavy chain (Mr of 56,000) or of two IgG heavy chains and one molecule of the receptor chain. The presence of this protein in control samples was not observed at all or its content was markedly lower. The same effect was also observed when DTBP, a cleavable crosslinking reagent was used. In this case, 125I-IgG heavy and light chains or the receptor subunit chains were found after SDS-PAGE under reducing conditions. The results presented in this paper do not suggest a presence of additional chains in the placental Fc gamma receptor others than described in our previous paper concerning the subunit structure of this receptor.

Cross-Linking Reagents↗
PubMed1978

Primary and secondary tryptic cleavages of human IgM at high temperature.

Previously it was shown that the tryptic digestion of human IgM at 65 C yield Fc'mu fragments in addition to Fabmu and (Fc)5mu fragments. This Fc'mu was found to be derived from the Cmu4 domain of the mu-chain. Additional studies were done on the effects of tryptic digestion time and temperature on the proteolytic process of IgM, IgM digestion by 2% trypsin at 65 C produced (Fc)8mu and Fc'mu in approximately a 3:2 ratio. While an appreciable amount of intact (Fc)5mu was present along with Fc'mu in the IgM digest, no residual "(Fc)5mu fragment" with its C-terminal segment missing was found. If the temperature of digestion is held constant at either 56 C or 60 C and the digestion time is varied from 20 to 90 min, there is also progressive cleavage of IgM with a concomitant increase in the yield of Fc'mu. It appears that the tryptic digestion of IgM is a stepwise process and that the primary cleavage of IgM occurs at Arg-325(2) of all ten mu-chains and the (Fc)5mu is subsequently degraded to Fc'mu fragments by secondary cleavage.

Alkylation↗
PubMed1988

Characterization of type I collagen from the skin of blue grenadier (Macruronus novaezelandiae).

The skin collagen of a fish, blue grenadier (Macruronus novaezelandiae), has been purified and characterized. The fish skin was readily soluble in dilute acetic acid, with no pepsin treatment needed. The collagen was purified by salt precipitation. Skin samples from fish of various ages showed that even in the oldest sample, more than 8 years of age, the collagen was still readily acid soluble. The purified collagen had a melting temperature of 22 degrees C; the shrinkage temperature for the skin was 48 degrees C. Its tissue distribution, examined by immunohistology, and its chemical properties indicated a close homology to mammalian type I collagen. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that three distinct alpha-chains were present. These were purified by ion-exchange chromatography on CM-cellulose and by gel permeation chromatography on Superose 6. The three purified alpha-chain fractions were examined by amino acid analysis and by SDS-PAGE of their cyanogen bromide fragments. These data indicated that the additional chain was genetically distinct, and most closely related to the alpha 1-chain, from which it was poorly resolved on SDS-PAGE.

Age Factors↗
PubMed1988

Generation of superoxide anion and lipid peroxidation in different cell types and subcellular fractions from rat testis.

Mitochondria and microsomes from whole rat testis, seminiferous tubules and Leydig cells were investigated with respect to their capacity to generate superoxide anion. In addition, lipid peroxidation by whole testis mitochondria and microsomes was measured. In the presence of NADH and various respiratory inhibitors all three mitochondrial preparations catalyzed the formation of superoxide anion at a rate of 0.27-1.67 nmol/min.mg. This formation was concluded to be confined mainly to the NADH dehydrogenase region of the respiratory chain. Addition of NADPH to whole testis or Leydig cell mitochondria, but not tubule mitochondria, caused an additional formation of superoxide anion, which was unrelated to the respiratory chain, accelerated several-fold by menadione, and presumably catalyzed by NADPH-cytochrome c reductase and cytochrome P-450. Microsomes isolated from whole testis, seminiferous tubules, and Leydig cells generated superoxide anion at rates between 0.19 and 0.44 nmol/min.mg. These rates were also strongly stimulated by menadione. It is likely that both NADPH-cytochrome c reductase and cytochrome P-450 were involved in the microsomal generation of superoxide. Free radical scavengers of various types inhibited both the mitochondrial and microsomal formation of superoxide anion. Lipid peroxidation in whole testis essentially paralleled superoxide anion generation. However, the rate of mitochondrial lipid peroxidation was twice that of the microsomal rate. It is concluded that seminiferous tubules and Leydig cells generate superoxide anion at different rates and by different mechanisms. Together with cytochrome P-450-dependent hydroxylases, e.g., BP and DMBA hydroxylases, this superoxide generation may reflect a potential for cell-specific peroxidative damage in the testis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2002

Lewis acid-promoted Kharasch-Curran additions: a competition kinetics study of bromine atom transfer addition of N-alpha-bromoacetyl-oxazolidinone to 1-hexene.

Lewis acids can efficiently promote free radical atom transfer reactions of an oxazolidinone imide substrate, 1, derived from alpha-bromo acetic acid. Thus, 1 undergoes a radical chain addition to 1-hexene giving the atom transfer addition compound, 6, in the presence of scandium or ytterbium triflate in 1,2-dichloroethane or a cosolvent mixture of 1/9 THF/dichloromethane. In 1,2-dichloroethane the solution is heterogeneous, while the cosolvent mixture gives a homogeneous solution, even at temperatures of -78 degrees C. Competition experiments were carried out in both solvent systems with added carbon tetrachloride to study how Lewis acid affected the product distribution. In the presence of carbon tetrachloride, chloride 7 is formed in addition to 6 and the ratio of these two products depends on the amount of Lewis acid present. In the presence of ytterbium triflate, in the cosolvent system, the reaction rate of bromine atom transfer was enhanced up to 400-fold compared to the reaction without added Lewis acid. Significant rate enhancements were also obtained in the solvent 1,2-dichloroethane, although the analysis of the system is complicated by the heterogeneous nature of the medium. Computation of C-Br bond dissociation energies (BDE) of the complexed and uncomplexed oxazolidinone bromide suggest that complexation lowers the BDE due to the effect of the strong electron-withdrawing group on the C-Br bond dipole.

Alkenes↗
PubMed1981

[Tunicamycin inhibition of carbohydrate incorporation into thyroglobulin].

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.

Animals↗
PubMed2003

Ab initio structure prediction of two alpha-helical oligomers with a multiple-chain united-residue force field and global search.

A hierarchical methodology for ab initio structure prediction is extended to treat oligomeric proteins. Modifications are made to a united-residue (UNRES) force field and a Conformational Space Annealing (CSA) global search method. The computational cost of including additional chains and the increase in speed from symmetry optimizations are evaluated. The native structures of two oligomeric proteins from the CASP3 exercise, the retro-GCN4 leucine zipper and the synthetic domain-swapped dimer, were identified as the lowest-energy families resulting from the search of the proteins when rotational symmetry was imposed. Additional searches in different symmetries and oligomerization states were carried out, and the results indicate some problems in the thoroughness of the search and in the search of packing arrangements if symmetry constraints are not imposed.

Leucine Zippers↗
PubMed1985

A heterozygous collagen defect in a variant of the Ehlers-Danlos syndrome type VII. Evidence for a deleted amino-telopeptide domain in the pro-alpha 2(I) chain.

A structural defect in the alpha 2(I) chain of type I collagen was characterized in a new case of the Ehlers-Danlos syndrome type VII. The patient's skin, fascia, and bone collagens all showed an abnormal additional chain, pN-alpha 2(I)s, running slower than the alpha 2(I) chain on electrophoresis. The extension was shown to be on the amino-terminal fragment of pN-alpha (I)s by cleavage with human collagenase, but pepsin was unable to convert pN-alpha 2(I)s to alpha 2(I). Skin collagen was 4-fold more extractable and contained fewer beta-dimers and a lower concentration of cross-linking amino acids than control skin collagen. Electron micrographs of both dermis and bone showed markedly irregular ragged outlines of the collagen fibrils in cross-section, although the patient had no clinical signs of bone disease. Procollagen secreted by her skin fibroblasts in culture showed equal amounts of the normal and abnormal alpha 2(I) chains on pepsin digestion. Before pepsin, the pN-alpha 2(I) component ran as a doublet on electrophoresis; pepsin removed only the normal slower chain. The suspected deletion in pN-alpha 2(I)s was traced by CNBr peptide analysis to the N-propeptide fragment, which behaved on electrophoresis about 15-20 residues smaller than that from the normal pN-alpha 2(I) chain. The simplest genetic explanation is a spontaneous heterozygote in which one normal and one abnormal allele for the pro-alpha 2(I) gene are expressed, the protein defect being a deletion of the junction domain that spans the N-propeptidase cleavage site and the N-telopeptide cross-linking sequence.

Bone and Bones↗
PubMed1984

Hemoglobin of the adult white stork (Ciconia ciconia, ciconiiformes). The primary structure of alpha A- and beta-chains from the only present hemoglobin component.

The hemolysate obtained from erythrocytes of the adult White Stork (Ciconia ciconia) contains only one hemoglobin component, identified to be HbA. The complete primary structures of alpha A- and beta-chains are presented. The minor hemoglobin component HbD with alpha D-chains usually present in adult avian species was not detected by the White Stork. The sequence was determined by automatic Edman degradation of tryptic peptides and in the case of beta-chains additionally of the C-terminal peptide obtained by chemical cleavage at the Asp-Pro bond. Homologous comparison with the Greylag Goose (Anser anser) hemoglobin showed that the alpha A-chains differ by 23 amino-acid exchanges, the beta-chains by 17. Four of the substitutions in the alpha A-chains are in the alpha 1 beta 1-contact points, one in the alpha 1 beta 2-contacts and one in the amino acids near the heme. The amino-acid substitutions of the White Stork hemoglobin as compared to the other avian hemoglobins are discussed. We suggest that alpha D-chain is persistence of an embryonic gene.

Amino Acid Sequence↗
PubMed1994

Ca(2+)-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells.

Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca2+ concentration required for half-maximal phosphorylation was 500 nM for MLCK and 250 nM for myosin light chain. Addition of KN-62 or a synthetic peptide CK II, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase II activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodamine-calmodulin in intact tracheal smooth muscle cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗