Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase.

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.

Calsequestrin, a component of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store of chicken cerebellum.

The presence and distribution of calsequestrin (CS), Ca2+ pump, and inositol 1,4,5-trisphosphate (IP3) receptor were investigated biochemically and immunologically in microsomal (P3) fractions isolated from chicken cerebrum and cerebellum. Two different batches of polyclonal antibodies specific for chicken skeletal muscle CS identified a Ca2+ binding, CS-like protein that was extremely enriched in cerebellum P3 fractions and absent from all cerebrum fractions. The cerebellum CS-like protein was deemed authentic CS because the N-terminal amino acid domain and peptide mapping were identical to those of skeletal muscle CS in the same species. CS was detected in striated muscles and cerebellum only. Cerebellum P3 fractions were also found to be considerably enriched in Ca2+ pump and IP3 receptor compared with the homologous cerebrum fractions, as judged by measurements of Ca2+ uptake, Ca2(+)-ATPase activity, IP3-induced Ca2+ release, and [3H]IP3 binding, respectively. Cerebellum microsomal fractions therefore appear to contain membrane fragments endowed with Ca2+ pump, IP3 receptor, and CS, i.e., three key components of a Ca2+ storage organelle.

Chemical modification of the Ca(2+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum: identification of sites labeled with aryl isothiocyanates and thiol-directed conformational probes.

The Ca(2+)-ATPase protein of rabbit skeletal muscle sarcoplasmic reticulum is a single polypeptide chain of 1001 amino-acid residues. Among these residues are 24 Cys, 9 of which have previously been shown to be accessible to one or more thiol-specific reagents. Many studies on the structure and function of this Ca(2+)-ATPase have made use of sulfhydryl-directed, conformationally-sensitive probes, but the labeling sites for these probes have been directly identified in only a few cases, causing uncertainty in the interpretation of results. In the present work, we have investigated the Ca(2+)-ATPase labeling sites for three thiol-directed spectroscopic probes: fluorescein 5'-maleimide (Fmal), 4-dimethylaminophenyl-azo phenyl-4'-maleimide (DABmal), and 4-dimethylaminophenylazophenyl-4'-iodoacetamide (DABIA). Labeled Ca(2+)-ATPase was digested exhaustively with trypsin, and labeled peptides were purified and sequenced in order to identify the labeled Cys residues. Our results do not support the widely held assumptions that Cys-344 and Cys-364 are the most reactive residues with maleimide-based reagents, while Cys-670 and Cys-674 react most rapidly with iodoacetamide derivatives. We found instead that Fmal reacted most rapidly with Cys-471, followed by Cys-364, and more slowly with Cys-498, -525, -614 and -636. DABmal reacted most rapidly with Cys-364, followed by Cys-614, and more slowly with Cys-471, -498, -636 and -670. Cys-344 was not labeled by either Fmal or DABmal. DABIA reacted with the same six Cys residues, including Cys-670, as were labeled with DABmal, but in much lower yield. There was no evidence for labeling of Cys-674 with DABIA. The high reactivity of Fmal, but not the more hydrophobic DABmal, with Cys-471 is of interest because of previous studies suggesting that the accessibility of Cys-471 is influenced by ATP and that fluorescein derivatives bind to a hydrophobic pocket in the ATP binding site. Another derivative, fluorescein-5'-isothiocyanate (FITC), is thought to label the catalytic site of the Ca(2+)-ATPase and has been widely used as a conformational probe in structure-function studies on this and related proteins. We reinvestigated the chemical modification of the Ca(2+)-ATPase by FITC and 4-dimethyl-aminophenyl-4'-isothiocyanate (DABITC). Incorporation of stoichiometric amounts of FITC resulted in a nearly complete loss of ATPase activity. Labeling and inactivation of the Ca(2+)-ATPase by FITC did not occur in the presence of ATP. DABITC was less reactive than FITC, and did not inactivate the Ca(2+)-ATPase to any significant extent.(ABSTRACT TRUNCATED AT 400 WORDS)

Tryptic and chymotryptic cleavage sites in sequence of alpha-subunit of (Na+ + K+)-ATPase from outer medulla of mammalian kidney.

Localization of selective proteolytic splits in alpha-subunit of (Na+ + K+)-ATPase is important for understanding the mechanism of active Na+,K+-transport. Proteolytic fragments of alpha-subunit from pig kidney were purified by chromatography in NaDodSO4 on TSK 3000 SW columns. NH2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of alpha-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691-695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490-1495). The primary chymotryptic split in the E1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E1-form is located between Lys-30 and Glu-31 (Bond 2). In the E2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH2-terminal for E1-E2 transition. Protonation of His-13 facilitates transition from E1- to E2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na+ and the shift in poise of E1-E2 equilibrium in direction of E1-forms. The NH2-terminal sequence in renal alpha-subunit is not conserved in alpha + from rat neurolemma or in alpha-subunit from Torpedo or brine shrimp. A regulatory function of the NH2-terminal part of the alpha-subunit may thus be a unique feature of the alpha-subunit in (Na+ + K+)-ATPase from mammalian kidney.

Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.

In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.

Interaction of a troponin I inhibitory peptide with both domains of troponin C.

Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC.

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C.

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.

A calmodulin endoproteinase from mitochondrial membranes.

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.

Calcium-induced flexibility changes in the troponin C-troponin I complex.

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.

Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain.

Highly purified lamb kidney (Na+ + K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na+ + K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (gamma 1 and gamma 2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that gamma 1 is an aggregated form of gamma 2. The total yield was 0.8-1.5 mol of gamma component per mol of catalytic subunit recovered. This indicates that the gamma component is present in stoichiometric amounts in the Na+ + K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.

Tryptic digest of the alpha subunit of lamb kidney (Na+ + K+)-ATPase.

The Mr approximately equal to 100 000 alpha subunit was prepared from highly purified lamb kidney (Na+ + K+)-ATPase. Its N-terminal sequence is Gly-Arg-Asx-Lys-Tyr-Glu. The alpha subunit was S-carboxymethylated, succinylated, and cleaved at its 40 arginine residues with trypsin. Four major, well-differentiated peptide fractions (A to D) were obtained by chromatography of the digest on a Sephadex G-50 column. Fraction A eluted at the void volume of the column and contained aggregated, very hydrophobic peptides, possibly from regions of alpha that are buried within the membrane lipid bilayer in the native enzyme. Fractions B to D, which together accounted for about 75% of the total protein, contained water-soluble peptides. To test the feasibility of using antibodies to identify and purify specific peptides of alpha subunit, studies were carried out using antibodies to native (Na+ + K+)-ATPase. Carboxymethylation and succinylation did not significantly decrease total antibody binding to alpha subunit, although the affinity of the anti-(Na+ + K+)-ATPase antibodies for alpha subunit was reduced by about 50%. The tryptic peptides of alpha subunit also retain significant immunochemical reactivity. Fractions A, B and C (but not D) of the digest all bind antibodies. To characterize further the tryptic digest, 16 peptides from fraction D were isolated and sequence studies on these were carried out.

Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain.

The proteinase previously described as an unidentified component of E. coli A2 extracts which hydrolyses actin at a new cleavage site (Khaitlina et al. (1991) FEBS Lett. 279, 49) was isolated and further characterized. A chromatographic method of proteinase purification was developed by which a purity of more than 80% was attained. The enzyme was identified as a single, 32 kDa polypeptide (ECP 32) by SDS-PAGE and non-denaturing electrophoresis as well as by ion-exchange chromatography and gel filtration. The N-terminal sequence of ECP 32 was determined to be: AKTSSAGVVIRDIFL. The activity of ECP 32 is inhibited by o-phenanthroline, EDTA, EGTA and zincone. The EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions. Based on these properties ECP 32 was classified as a metalloproteinase (EC 3.4.24). Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 was observed at enzyme substrate mass ratios of 1:25 to 1:3000. Two more sites between Ala-29 and Val-30, and between Ser-33 and Ile-34 were cleaved by ECP 32 in heat- or EDTA-inactivated actin. Besides actin, only histones and DNA-binding protein HU were found to be substrates of the proteinase, confirming its high substrate specificity. Its molecular mass, N-terminal sequence and enzymatic properties distinguish ECP 32 from any known metalloproteinases of E. coli, and we therefore conclude that it is a new enzyme.

Effects of flux enhancing polymer on the characteristics of sludge in membrane bioreactor process.

A newly developed membrane performance enhancer (MPE) was used to prevent membrane fouling in a membrane bioreactor (MBR) process. It transpired that 1,000 mg/l of MPE reduced polysaccharide levels from 41 mg/I to 21 mg/I on average under the experimental condition. Repeated experiments also confirmed that 50-1,000 mg/l of MPE could reduce membrane fouling significantly and increase the intervals between membrane cleanings. Depending on MPE dosages and experimental conditions, trans-membrane pressure (TMP) increase was suppressed for 20-30 days, while baseline TMP surged within a few days. In addition, MPE allowed MBR operation even at 50,000 mg/l of total solid and reduced permeate COD. However, no evidence of toxicity for sludge was found from respiratory works.

Changes in adrenocorticotropin and cortisol responsiveness after repeated partial umbilical cord occlusions in the late gestation ovine fetus.

Despite many studies reporting fetal ACTH and cortisol (F) responses to acute fetal hypoxemia induced by several methods, effects of repeated short-term fetal hypoxia produced by umbilical cord occlusion (UCO) on ACTH and F are unknown. We examined fetal ACTH and F responses to repeated, controlled, 50% reductions in common umbilical arterial blood flow (CUBF) produced by an inflatable cord occluder. Ten sheep fetuses were instrumented at 123-128 days gestation (dGA) with arterial, venous, and amniotic catheters. A common umbilical artery transit-time ultrasound flow probe was implanted to measure CUBF. An inflatable occluder was placed around the proximal portion of the umbilicus. In five fetuses (group I) at 131 +/- 1 dGA (mean +/- SEM), 12 UCOs (CUBF reduced by 50%), each lasting 5 min separated by 15 min recovery, were performed. Changes in fetal arterial blood gases, pH and plasma ACTH, and F concentrations were determined before, during, and after the 1st, 6th, and 12th UCOs. Sham experiments were conducted on the other five fetuses at 130 +/- 1 dGA (group II). In group I, CUBF decreased to 49 +/- 1% (mean +/- SEM of 12 UCOs). After each UCO, CUBF returned to baseline within 5 min. A modest fall in fetal arterial PO2 and arterial pH (21.2 +/- 0.2 to 16.8 +/- 0.2 mmHg and 7.33 +/- 0 to 7.29 +/- 0, respectively) and a mild increase in fetal PaCO2 (49.9 +/- 0.5 to 54.9 +/- 0.4 mmHg; mean +/- SEM of 12 UCOs) occurred with each UCO. Whereas preocclusion fetal ACTH concentrations increased by the 12th UCO, F remained unchanged. Fetal ACTH increased after the 1st, 6th, and 12th UCOs. Fetal F increased after the 1st and 6th UCOs but not after the 12th UCO. Fetal plasma ACTH and F remained unchanged throughout the experiments in group II fetuses. We conclude that: 1) partial reductions in CUBF induce significant activation of the fetal anterior pituitary-adrenocortical axis in late-gestation fetal sheep; 2) after repeated UCOs, fetal ACTH responsiveness is maintained, but fetal F responses become attenuated.

Phosphorylation of brush border myosin by brush border calmodulin-dependent myosin heavy and light chain kinases.

Calmodulin-dependent myosin light chain kinase isolated from chicken intestinal brush border phosphorylates brush border myosin at an apparently single serine identical to that phosphorylated by smooth muscle myosin light chain kinase. Phosphorylation to 1.8 mol phosphate/mol myosin activated the myosin actin-activated ATPase about 10-fold, to about 50 nmol/min per mg. Myosin phosphorylated on its light chains could then be further phosphorylated to a total of 3.2 mol phosphate per mol by brush border calmodulin-dependent heavy chain kinase. Heavy chain phosphorylation did not alter the actin-activated ATPase of either myosin prephosphorylated on its light chains or of unphosphorylated myosin.

Brush border myosin heavy chain phosphorylation is regulated by calcium and calmodulin.

Myosin from chicken intestinal brush borders is phosphorylated on its heavy chains at threonine by a kinase isolated from brush borders. In contrast to other heavy chain kinases, the brush border kinase activity is dependent on calcium and calmodulin. The partially purified preparation also phosphorylated myosin on its light chains at serine, but in a calmodulin-independent manner. Phosphorylation of the light chains in the absence of calmodulin or both heavy and light chains in the presence of calmodulin activated its actin-activated ATPase activity about 10-fold, to about 50 nmol/min per mg.

Phosphorylation of brush border myosin at threonine on its 20 kDa light chains by a calmodulin-independent kinase activates its ATPase.

A calmodulin-independent kinase isolated from chicken intestinal brush border phosphorylates brush border myosin mainly at an apparently single threonine on its 20 kDa light chains. Phosphorylation to 1.9 mol phosphate/mol myosin activated the myosin actin-activated ATPase about 12-fold, to about 100 nmol/min per mg. Brush border myosin ATPase can thus be activated by phosphorylation either at threonine, by calmodulin-independent kinase, or at serine, by calmodulin-dependent myosin light chain kinase, as previously shown [(1987) FEBS Lett. 223, 262-266].

Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon.

Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with Mr 25-27 kDa or in the short N-terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153. The primary structure of the tryptic peptide containing the main site of duck gizzard caldesmon phosphorylation is S-E-V-N-A-Q-N-X-V-A-E-D-E-T-K, where X is an unidentified residue, presumed to be phosphoserine. Thus, Ser73 is the main site phosphorylated by casein kinase II in avian gizzard caldesmon.

The localization of the cAMP-dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B.

Rap 1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap 1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxy terminus and is adjacent to the site of membrane attachment of the protein.

Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle.

A caldesmon kinase activity was detected in an ATP extract of the myofibril-like pellet from sheep aorta. The enzyme was purified 745-fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon-h. Ser-73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.