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P K Ngai

Publications and source records attributed to P K Ngai.

18 recordsLinked to original sources

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase.

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, alpha-actin-in and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryo-specific isoform with a different molecular mass was detected.

Amino Acid Sequence↗

Protein A antibody-capture ELISA (PACE): an ELISA format to avoid denaturation of surface-adsorbed antigens.

Adsorption to a polymeric surface may severely alter the antigenic structure of proteins through unfolding. A conventional capture ELISA in which a protein antigen is adsorbed to the microtiter plate may be unsuitable for testing the specificity of antibodies directed against native proteins (C. Schwab and H.R. Bosshard (1992) J. Immunol. Methods 147, 125). This problem can be overcome by PACE, a new ELISA procedure in which monoclonal or polyclonal antibodies are first allowed to equilibrate with biotinylated antigen in solution. Thereafter, the antigen-antibody complex (and free antibody) is bound to the microtiter plate through protein A. Captured antigen-antibody complex is detected by streptavidin-alkaline phosphatase and p-nitrophenylphosphate. A competition assay is accomplished by co-incubation of biotinylated and non-biotinylated antigens before capture to the protein A-coated plate. PACE combines the advantages of a solution-phase immunoassay (Farr assay) with the ease of a solid-phase ELISA. PACE has been used to test the conformational specificity of polyclonal and monoclonal antibodies against native and denatured cytochrome c, and of a polyclonal antiserum against a coiled coil leucine zipper peptide. Since a biotin group can be attached specifically to the N-terminal residue of synthetic peptides, PACE is also useful for assaying reactivity against peptide antigens which are difficult to adsorb to microtiter plates.

Amino Acid Sequence↗

Relatedness of a periplasmic, broad-specificity RNase from Aeromonas hydrophila to RNase I of Escherichia coli and to a family of eukaryotic RNases.

The isolation, sequencing, and characterization of a periplasmic RNase gene from Aeromonas hydrophila AH1133 is described. Following subcloning of the gene on a 2.7-kb PstI fragment, its direction of transcription and approximate location were determined. Analysis of the nucleotide sequence reveals that the gene is 645 bp long, coding for 215 amino acid residues with a total molecular weight of 24,215. A typical leader sequence is present at the beginning of the corresponding protein. Computer analysis revealed strong local similarities to Escherichia coli RNase I and to the active site of a family of eukaryotic RNases. Expression studies indicate that the RNase natural promoter functions poorly in E. coli. In this organism, the enzyme is mainly localized in the cytoplasm and periplasm, although high levels of expression lead to significant release into the extracellular medium. Functional and physical characterizations further indicate that the periplasmic and cytoplasmic enzymes of A. hydrophila are likely to be the counterparts of E. coli RNase I and its cytoplasmic form RNase I*: as for the E. coli enzymes, the A. hydrophila RNase forms have similar sizes and show broad specificity, and the periplasmic form is more active towards natural polymer RNA than its cytoplasmic counterpart. Both forms are relatively thermosensitive and are reversibly inactivated by up to 0.6% sodium dodecyl sulfate. Southern hybridization revealed homology to E. coli K-12 and Shigella sp. genomic DNA, a finding which correlates with the presence of secreted RNases in these organisms. In contrast, species of phylogenetically closer genera, such as Vibrio and Plesiomonas, did not hybridize to the A. hydrophila RNase gene.

Aeromonas hydrophila↗

Identification of a reactive lysyl residue (Lys103) of recombinant human interleukin-1 beta. Mechanism of its reactivity and implication of its functional role in receptor binding.

Recombinant human interleukin-1 beta (h-IL-1 beta) was chemically modified with 4-(N,N-dimethylamino)-4'-isothiocyanatoazobenzene-2'-sulfonic acid (S-DABITC), a water-soluble color reagent specific for lysine labeling. Modified h-IL-1 beta was digested by lysyl endopeptidase. Peptides containing labeled lysines were detected at the visible wavelength (436 nm) and isolated by HPLC. The modification sites were eventually determined by sequence analysis. The results revealed that Lys103, Lys92, Lys93, and Lys94 of h-IL-1 beta reacted selectively with S-DABITC. A 1-h incubation with 1 mM S-DABITC at room temperature resulted in a quantitative modification of Lys103, 22% of Lys92, 27% of Lys93, and 18% of Lys94, respectively. This modification was accompanied by a 20-fold decrease of the protein's ability to bind to the receptor. Furthermore, a mutant of h-IL-1 beta (M9, Glu105 substituted by Lys) exhibits markedly impaired receptor binding, and the S-DABITC reactivity of its Lys103 was found to be reduced by 90%. These findings suggest that Lys103 of h-IL-1 beta might play an important role in the h-IL-1 beta/receptor interaction.

Amino Acid Sequence↗

A novel one-step purification of human alpha-thrombin after direct activation of crude prothrombin enriched from plasma.

Crude prothrombin enriched from human plasma was directly activated to generate alpha-thrombin without prior exhaustive purification of the proenzyme using a combination of several different types of chromatographic techniques, as in all previously described methodologies. Activated thrombin was separated from other components in a single step by taking advantage of its highly specific affinity to heparin immobilized on a matrix support of Sepharose CL-6B. On the basis of the data presented herein, we have demonstrated the ease with which at least 25 mg of a highly purified enzyme (greater than 97% homogeneous by laser densitometry) can be obtained per litre of plasma. Our product exhibits a specific activity of at least 4000 National Institutes of Health units/mg and is stable after being freeze-dried for the purpose of long-term storage.

Amino Acids↗

The structural elements of hirudin which bind to the fibrinogen recognition site of thrombin are exclusively located within its acidic C-terminal tail.

Six lysyl residues of human thrombin (LysB21, LysB52, LysB65, LysB106, LysB107 and LysB154) have been previously shown to participate in the binding site of hirudin, a thrombin-specific inhibitor [(1989) J. Biol. Chem. 264, 7141-7146]. In this report, we attempted to delineate the region of hirudin which binds to these basic amino acids of thrombin. Using the N-terminal core domains (r-Hir1-43 and r-Hir1-52) derived from recombinant hirudins and synthetic C-terminal peptides (Hir40-65 and Hir52-65)--all fragments form complexes with thrombin--we are able to demonstrate that the structural elements of hirudin which account for the shielding of these 6 lysyl residues are exclusively located within the acidic C-terminal region. Since hirudin C-terminal peptides were shown to bind to a non-catalytic site of thrombin and inhibit its interaction with fibrinogen [(1987) FEBS Lett. 211, 10-16], our data consequently imply that these 6 lysyl residues are constituents of the fibrinogen recognition site of thrombin.

Amino Acid Sequence↗

Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.

Actins↗

The effects of phosphorylation of smooth-muscle caldesmon.

Caldesmon is a major calmodulin- and actin-binding protein of smooth muscle which interacts with calmodulin in a Ca2+-dependent manner or with actin in a Ca2+-independent manner. Isolated caldesmon is capable of inhibiting the actin-activated Mg2+-ATPase of smooth-muscle myosin, suggesting a possible physiological role for caldesmon in regulating the contractile state of smooth-muscle. Caldesmon can be phosphorylated in vitro by a co-purifying Ca2+/calmodulin-dependent protein kinase and dephosphorylated by a protein phosphatase, both of which are present in smooth muscle. We investigated further the phosphorylation of caldesmon and the effects which phosphorylation has on the functional properties of the protein. The kinetics of caldesmon phosphorylation were similar whether the caldesmon substrate was free or bound to actin, actin/tropomyosin or thin filaments. Caldesmon containing endogenous kinase activity was rapidly phosphorylated (to approx. 1 mol of Pi/mol of caldesmon in 5 min) when reconstituted with actin, myosin, tropomyosin, calmodulin and myosin light-chain kinase in the presence of Ca2+ and MgATP2-. Under conditions in which unphosphorylated caldesmon showed substantial inhibition of the actin-activated myosin Mg2+-ATPase, no inhibition was observed with phosphorylated caldesmon. This was the case whether caldesmon was phosphorylated before addition to the actomyosin Mg2+-ATPase system, or phosphorylation was allowed to take place during the ATPase reaction. Binding studies revealed maximal binding of 1 mol of unphosphorylated caldesmon/9.5 mol of actin and 1 mol of phosphorylated caldesmon/11.7 mol of actin. All the bound phosphorylated caldesmon could be released by Ca2+/calmodulin, with half-maximal release at 0.11 microM-Ca2+, whereas only 62% of the bound unphosphorylated caldesmon could be removed, with half-maximal release at 0.16 microM-Ca2+. However, under conditions in which inhibition of actomyosin Mg2+-ATPase activity by non-phosphorylated but not by phosphorylated caldesmon was observed, both forms of caldesmon would remain bound to the thin filament. These observations suggest a possible mechanism whereby caldesmon phosphorylation may prevent its inhibitory action on the actomyosin Mg2+-ATPase.

Actins↗

Activation of smooth muscle myosin Mg2+-ATPase by native thin filaments and actin/tropomyosin.

Application of the myosin competition test (Lehman, W., and Szent-Györgyi, A. G. (1975) J. Gen. Physiol. 66, 1-30) to chicken gizzard actomyosin indicated that this smooth muscle contains a thin filament-linked regulatory mechanism. Chicken gizzard thin filaments, isolated as described previously (Marston, S. B., and Lehman, W. (1985) Biochem. J. 231, 517-522), consisted almost exclusively of actin, tropomyosin, caldesmon, and an unidentified 32-kilodalton polypeptide in molar ratios of 1:1/6:1/26:1/17, respectively. When reconstituted with phosphorylated gizzard myosin, these thin filaments conferred Ca2+ sensitivity (67.8 +/- 2.1%; n = 5) on the myosin Mg2+-ATPase. On the other hand, no Ca2+ sensitivity of the myosin Mg2+-ATPase was observed when purified gizzard actin or actin plus tropomyosin was reconstituted with phosphorylated gizzard myosin. Native thin filaments were rendered essentially free of caldesmon and the 32-kilodalton polypeptide by extraction with 25 mM MgCl2. When reconstituted with phosphorylated gizzard myosin, caldesmon-free thin filaments and native thin filaments exhibited approximately the same Ca2+ sensitivity (45.1 and 42.7%, respectively). The observed Ca2+ sensitivity appears, therefore, not to be due to caldesmon. Only trace amounts of two Ca2+-binding proteins could be detected in native thin filaments. These were identified as calmodulin (present at a molar ratio to actin of 1:733) and the 20-kilodalton light chain of myosin (present at a molar ratio to actin of 1:270). The Ca2+ sensitivity observed in an in vitro system reconstituted from gizzard thin filaments and either skeletal myosin or phosphorylated gizzard myosin is due, therefore, to calmodulin and/or an unidentified minor protein component of the thin filaments which may be an actin-binding protein involved in regulating actin filament structure in a Ca2+-dependent manner.

Actins↗

Ca2+- and calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin.

Fodrin, a spectrin-like actin and calmodulin binding protein, was purified to electrophoretic homogeneity from a membrane fraction of bovine brain. The effect of fodrin on smooth muscle actomyosin Mg2+-ATPase activity was examined by using a system reconstituted from skeletal muscle actin and smooth muscle myosin and regulatory proteins. The simulation of actomyosin Mg2+-ATPase by fodrin showed a biphasic dependence on fodrin concentration and on the time of actin and myosin preincubation at 30 degrees C. Maximal stimulation (50-70%) was obtained at 3 nM fodrin following 10 min of preincubation of actin and myosin. This stimulation was also dependent on the presence of tropomyosin. In the absence of myosin light chain kinase, the fodrin stimulation of Mg2+-ATPase could not be demonstrated with normal actomyosin but could be demonstrated with acto-thiophosphorylated myosin, suggesting that fodrin stimulation depends on the phosphorylation of myosin. Fodrin stimulation was shown to require the presence of both Ca2+ and calmodulin when acto-thiophosphorylated myosin was used. These observations suggest a possible functional role of fodrin in the regulation of smooth muscle contraction and demonstrate an effect on Ca2+ and calmodulin on fodrin function.

Actins↗

Vascular smooth muscle caldesmon.

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.

Actins↗

Comparison of the effects of smooth and skeletal muscle actins on smooth muscle actomyosin Mg2+-ATPase.

Actin has been purified from smooth muscle (chicken gizzard) by two different procedures and its activation of smooth muscle myosin Mg2+-ATPase activity compared with that achieved with rabbit skeletal muscle actin. The procedure of Pardee and Spudich (Methods Enzymol. (1982) 85, 164-181) for the purification of rabbit skeletal muscle actin is readily applicable to the isolation of chicken gizzard actin, enabling large quantities to be purified in two days. Smooth muscle actin could be successfully stored as F-actin at -80 degrees C and survived freezing and thawing at least twice. Smooth muscle actin activated myosin Mg2+-ATPase to a higher level than its skeletal muscle counterpart (77.9 nmol Pi/min/mg myosin vs 48.1 nmol Pi/min/mg myosin).

Actins↗

Properties of caldesmon isolated from chicken gizzard.

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

Detection of caldesmon in muscle and non-muscle tissues of the chicken using polyclonal antibodies.

Polyclonal antibodies raised in rabbits against chicken gizzard caldesmon have been purified and used in immunoblotting experiments to study the distribution of this actin- and calmodulin-binding protein in diverse tissues of the chicken. Total homogenates and heat-treated homogenate supernatants derived from each tissue were subjected to sodium dodecyl sulfate-polyacrylamide gradient slab gel electrophoresis and immunoblotting using the horseradish peroxidase method. All chicken tissues examined contained caldesmon of Mr = 141,000. The amount of caldesmon found in the different tissues varied considerably and semi-quantitative comparison of stained immunoblots indicated the following relative caldesmon contents: gizzard greater than oesophagus greater than duodenum = small intestine greater than lung greater than aorta greater than heart = skeletal muscle greater than kidney = trachea greater than brain greater than liver. Each tissue revealed small amounts of lower Mr immunoreactive proteins, predominantly bands of Mr 94,000 and 70,000, which appear to be proteolytic fragments of caldesmon. Isolated caldesmon was found to be highly sensitive to proteolysis. The widespread distribution and similarity of caldesmon in different tissues of the chicken suggest its functional importance and structural conservation.

Animals↗

Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon.

Caldesmon, a major calmodulin- and actin-binding protein of smooth muscle (Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5652-5655), has been obtained in highly purified form from chicken gizzard by a modification of a previously published procedure (Ngai, P. K., Carruthers, C. A., and Walsh, M. P. (1984) Biochem. J. 218, 863-870) and was found to cause a significant inhibition of both superprecipitation and actin-activated myosin Mg2+-ATPase activity in a system reconstituted from the purified contractile and regulatory proteins without influencing the phosphorylation state of myosin. This inhibitory effect was seen both in the presence and absence of tropomyosin. A Ca2+-and calmodulin-dependent kinase which catalyzed phosphorylation of caldesmon was identified in chicken gizzard; this kinase is distinct from myosin light-chain kinase. Caldesmon prepared by calmodulin-Sepharose affinity chromatography was contaminated with caldesmon kinase activity and was unable to inhibit actomyosin ATPase activity or superprecipitation. Phosphatase activity capable of dephosphorylating caldesmon was also identified in smooth muscle. These results indicate that caldesmon can inhibit smooth muscle actomyosin ATPase activity in vitro, and this function may itself be subject to regulation by reversible phosphorylation of caldesmon.

Actins↗

Ca2+-dependent hydrophobic-interaction chromatography. Isolation of a novel Ca2+-binding protein and protein kinase C from bovine brain.

Several bovine brain proteins have been found to interact with a hydrophobic chromatography resin (phenyl-Sepharose CL-4B) in a Ca2+-dependent manner. These include calmodulin, the Ca2+/phospholipid-dependent protein kinase (protein kinase C) and a novel Ca2+-binding protein that has now been purified to electrophoretic homogeneity. This latter protein is acidic (pI 5.1) and, like calmodulin and some other high-affinity Ca2+-binding proteins, exhibits a Ca2+-dependent mobility shift on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with an apparent Mr of 22 000 in the absence of Ca2+ and Mr 21 000 in the presence of Ca2+. This novel calciprotein is distinct from known Ca2+-binding proteins on the basis of Mr under denaturing conditions, Cleveland peptide mapping and amino acid composition analysis. It may be a member of the calmodulin superfamily of Ca2+-binding proteins. This calciprotein does not activate two calmodulin-dependent enzymes, namely cyclic nucleotide phosphodiesterase and myosin light-chain kinase, nor does it have any effect on protein kinase C. It may be a Ca2+-dependent regulatory protein of an as-yet-undefined enzymic activity. The Ca2+/phospholipid-dependent protein kinase is also readily purified by Ca2+-dependent hydrophobic-interaction chromatography followed by ion-exchange chromatography, during which it is easily separated from calmodulin. A preparation of protein kinase C that lacks contaminating kinase or phosphatase activities is thereby obtained rapidly and simply. Such a preparation is ideal for the study of phosphorylation reactions catalysed in vitro by protein kinase C.

Amino Acids↗

Isolation of the native form of chicken gizzard myosin light-chain kinase.

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.

Animals↗