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PubMed1999

Regulation of fibrillin carboxy-terminal furin processing by N-glycosylation, and association of amino- and carboxy-terminal sequences.

The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12-15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.

Endoplasmic Reticulum↗
PubMed1997

Comparison of the clinical performance of the immunoenzymometric assays for N-terminal and C-terminal type I collagen telopeptides and the HPLC assay for pyridinium cross-links.

We evaluated the clinical performances of the immunoenzymometric assays for type I collagen N-terminal and C-terminal telopeptides and the HPLC assay for total deoxypyridinoline, in distinguishing between subjects with a moderately increased bone resorption rate (women in postmenopause) and subjects with normal bone resorption rate (women in premenopause). The postmenopausal group consisted of 61 women who had been in menopause for no more than 10 years, while the premenopausal group consisted of 52 healthy women with normal menstrual cycles. The biochemical markers were measured in a 24 hour urine sample and the results expressed as the molar ratio with urinary creatinine. The clinical performances were estimated by calculating the accuracy (as the area under a Receiver Operated Characteristic (ROC) curve: mean +/- SEM) and the discriminating power (as score) of each assay in distinguishing postmenopausal subjects from premenopausal subjects. Type I collagen C-terminal telopeptide, type I collagen N-terminal telopeptide and total deoxypyridinoline were significantly higher in the postmenopausal than in the premenopausal group (p < 0.01). Accuracies of these three markers ranged from 66.8 +/- 5.1% to 76.8 +/- 4.3%, while Z scores ranged from 3.54 to 5.67. Type I collagen C-terminal telopeptide, type I collagen N-terminal telopeptide and total deoxypyridinoline were not significantly different in their accuracy or discriminating power. All markers were highly correlated with coefficients of correlation ranging from 0.61 to 0.77. In summary, this study shows that 1) the immunoenzymometric assays for type I collagen N-terminal telopeptide and type I collagen C-terminal telopeptide show a high accuracy and discriminating power in distinguishing subjects with different bone resorption rate; 2) the results obtained with these immunoenzymometric assays are comparable to those obtained with the HPLC assay for total deoxypyridinoline. In conclusion our data support the use of the immunoenzymometric assays for type I collagen N-terminal telopeptide and type I collagen C-terminal telopeptide for estimating bone resorption.

Adult↗
PubMed1993

Individual and combined effects of intact PTH, amino-terminal, and a series of truncated carboxyl-terminal PTH fragments on alkaline phosphatase activity in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8.

The individual and combined effects of intact PTH, amino-terminal, and a series of truncated carboxyl-terminal PTH fragments on alkaline phosphatase activity were examined in dexamethasone-treated rat osteoblastic osteosarcoma cells ROS 17/2.8. Dexamethasone-induced alkaline phosphatase activity was inhibited not only by hPTH(1-84) and amino-terminal PTH fragment hPTH(1-34), but also by carboxyl-terminal PTH fragment hPTH(69-84) in a dose-related fashion. At 10(-7) mol/l, hPTH(1-84) completely abolished dexamethasone-induced alkaline phosphatase activity, while hPTH(1-34) and hPTH(69-84) reduced alkaline phosphatase activity to 0.16 +/- 0.02 and 0.80 +/- 0.03 fold, respectively, of the control value obtained in the absence of PTH peptides. The combination of hPTH(1-34) and hPTH(69-84) resulted in reduction of alkaline phosphatase activity to the level obtained by hPTH(1-84). The shorter carboxyl-terminal PTH fragment hPTH(71-84) did not affect alkaline phosphatase activity or modulate the action of hPTH(1-34). The longer carboxyl-terminal PTH fragment hPTH(53-84) stimulated alkaline phosphatase activity up to 1.23 +/- 0.03 fold and partially blunted the inhibitory effect of hPTH(1-34) on alkaline phosphatase activity. These findings suggest that carboxyl-terminal PTH fragments could exert diverse effects on the target cells, depending on the length of deletion of amino-terminal amino acids of PTH molecule, and interact with amino-terminal PTH fragment. The two amino-terminal amino acids of hPTH(69-84) and the 53-68 portion of hPTH(53-84) might be responsible for the respective inhibitory and stimulatory effects of the peptides on alkaline phosphatase activity.

Alkaline Phosphatase↗
PubMed1997

Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda.

The mechanisms that control N-protein-dependent antitermination in the phage lambda life cycle have counterparts in the regulatory systems of other organisms. Here we examine N-dependent antitermination at the intrinsic tR' terminator of lambda to elucidate the regulatory principles involved. The tR' terminator consists of a sequence of six base-pairs along the template at which the transcription complex is sufficiently destabilized to make RNA release possible. Within this "zone of opportunity" for termination the termination efficiency (TE) at each template position is determined by a kinetic competition between alternative reaction pathways that lead either to elongation or to termination. TE values at each position within tR' have been mapped as a function of NTP concentration, and it is shown that N protein (in the presence of NusA and a nut site; the minimal system for N-dependent antitermination) can offset increases in TE that are induced by limiting the concentrations of each of the next required NTPs. By limiting NTP concentrations or working at low temperature we show that a significant effect of N within the minimal system is to increase the rate of transcript elongation three- to fivefold at most positions along the template. Assuming that a comparable increase in elongation rate applies at template positions within the terminator, we show that an increase of this magnitude is not sufficient to account for the antitermination efficiency observed and that an approximately 100-fold stabilization of the transcription complex at intrinsic termination sites as a consequence of binding the N-containing antitermination sub-assembly must be invoked as well. A general method for partitioning TE effects in antitermination between changes in elongation rate and termination complex stability is demonstrated, based on competing free energy of activation barriers for the elongation and termination reactions. The analysis and utility of such mixed modes of transcriptional regulation are considered in general terms.

Bacterial Proteins↗
PubMed1987

Tau factor from Escherichia coli mediates accurate and efficient termination of transcription at the bacteriophage T3 early termination site in vitro.

The termination signal that limits transcription through the early region of bacteriophage T3 (T3Te) has been cloned and sequenced. The nucleotide sequence of T3Te is identical with that of T7Te, with the exception of a single G to U substitution in the 3' tail of the terminated transcript, and addition of an AC to the loop in the terminator stem-loop, enlarging the loop to six residues. Previous studies of the properties of T3Te have shown that this site is rho independent and is highly efficient for termination in vivo, but is used poorly in vitro during transcription with purified Escherichia coli RNA polymerase. In contrast, the equivalent site in bacteriophage T7 (T7Te) is an efficient termination signal both in vivo and in vitro. However, T3Te becomes an efficient termination site in vitro in the presence of preparations of tau factor. This factor also alters the sites of RNA chain termination found in vitro at T3Te. Transcripts formed in the presence of tau are several nucleotides shorter than those produced with RNA polymerase alone, and have 3' termini that are almost identical with transcripts found in vivo. These latter results are similar to our earlier findings with T7Te, and suggest that other rho independent terminators may act with transcription termination factors in vivo.

Base Sequence↗
PubMed1993

Characterization of the mouse beta maj globin transcription termination region: a spacing sequence is required between the poly(A) signal sequence and multiple downstream termination elements.

For the majority of mRNA encoding eukaryotic transcription units, there is little or no knowledge of the elements responsible for transcription termination or how they may interact with RNA polymerase. In this report, we have used recombinant adenovirus reporter vectors to characterize the mouse beta maj globin sequence elements that cause transcription termination. Within the globin 3' termination region, we have identified at least three sequence elements which induce significant levels of transcription termination (> 50%). The smallest functionally active element (64% termination) is 69 bp in length. The natural arrangement of these elements results in a cumulative termination which is greater than 90%. Recognition of the termination elements by RNA polymerase II depends on the presence of a functional poly(A) signal sequence. We demonstrate that efficient transcription termination depends on appropriate spacing between the poly(A) signal sequence and the termination element.

Animals↗
PubMed1995

Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of rhodanese.

A peptide consisting of the 17 N-terminal amino acids of native bovine rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.

Amino Acid Sequence↗
PubMed1998

Phi 29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates.

A 44 kDa C-terminal fragment of phi 29 DNA polymerase has been separately expressed and purified from Escherichia coli cells. As expected, the truncated protein lacked the 3'-5' exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of phi 29 DNA polymerase. On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn2+ as metal activator, although the catalytic efficiency was greatly reduced with respect to that of the complete enzyme. Moreover, and in contrast to the high processivity exhibited by phi 29 DNA polymerase (> 70 kb), polymerization by its C-terminal domain was completely distributive. All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization. Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of phi 29 DNA replication. We conclude that the C-terminal portion of phi 29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis. These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more specific functions (TP-priming, high processivity and strand-displacement) inherent to phi 29 DNA polymerase.

Bacillus Phages↗
PubMed1998

The C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase is required for efficient rho-dependent transcription termination.

We screened a collection of single alanine residue substitution mutants spanning the entire C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase (RNAP) for defects in rho-dependent transcription termination at lambdatR1 in vivo and in vitro, and thereby identified a patch of amino acid residues in the alphaCTD required for efficient rho-dependent termination. NusA addition led to the stimulation of rho-dependent termination under our conditions in vitro. The termination defects of a few mutant RNAPs could be attributed to altered interactions with the NusA protein, but rho-dependent termination by most of the defective RNAPs was still stimulated normally by NusA. The NusA-enhanced transcription pausing behaviors of the mutant RNAPs did not always correlate with their rho-dependent termination phenotypes. We conclude that the alphaCTD is a target for interactions with NusA that influence both termination and pausing, but in addition it participates in rho-dependent transcription termination in a NusA-independent manner.

Alanine↗
PubMed1990

Ultrastructural morphometric analysis of GABA-immunoreactive terminals in the ventrocaudal periaqueductal grey: analysis of the relationship of GABA terminals and the GABAA receptor to periaqueductal grey-raphe magnus projection neurons.

The periaqueductal grey (PAG) plays an important role in the descending modulation of nociception. Inhibitory influences of GABAergic terminals, located within the periaqueductal grey, are thought to play a role in antinociception by influencing the activity of neurons that project to the nucleus raphe magnus and adjacent reticular nuclei. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of GABA-immunoreactive terminals, and to visualize the neuronal distribution of the GABAA receptor in the ventrolateral periaqueductal grey of the rat. Of particular interest was a quantitative description of the interaction between GABA-immunoreactive axon terminals and periaqueductal grey neurons that were retrogradely-labelled from the nucleus raphe magnus and adjacent medullary reticular nuclei. Most terminals were observed to be immediately apposed only to two or three dendrites, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal grey, 37.5% of all GABA-immunoreactive terminals were adjacent to periaqueductal grey-nucleus raphe magnus and periaqueductal grey-reticular nucleus projection neurons. Symmetrical synapses with these retrogradely-labelled neurons were formed by 17% of GABA-immunoreactive terminals in the ventrolateral periaqueductal grey. We also noted that 13.2% of the GABA-immunoreactive terminals formed symmetrical synapses with GABA-immunoreactive dendrites in the periaqueductal grey, and occasionally those dendrites were retrogradely labelled. Only 0.8% of the GABA-immunoreactive terminals formed putative symmetrical synapses with other GABA-immunoreactive terminals. Consistent with these findings, GABAA receptor immunoreactivity was only associated with dendrites and perikarya in neurons of the ventrolateral PAG. These results are consistent with an inhibitory role for GABA on PAG neurons, a configuration required by hypothetical models for opoid disinhibitory circuitry within the PAG. In addition, the data further suggest that other kinds of GABAergic connections may be important in descending antinociception, and that a population of GABAergic PAG projection neurons exists that may be inhibitory within nucleus raphe magnus and the adjacent reticular nuclei.

Animals↗
PubMed1992

Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.

We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.

Amino Acid Sequence↗
PubMed2005

Consequences of C-terminal domains and N-terminal signal peptide deletions on LEKTI secretion, stability, and subcellular distribution.

The secretory lympho-epithelial Kazal-type-inhibitor (LEKTI) is synthesized as a pro-LEKTI protein containing an N-terminal signal peptide and 15 potentially inhibitory domains. This inhibitor is of special interest because of its pathophysiological importance for the severe congenital disease Netherton syndrome. We showed that LEKTI is a potent inhibitor of a family of serine proteinases involved in extracellular matrix remodeling and its expression is downregulated in head and neck squamous cell carcinomas. To assess the role of C-terminal domains and N-terminal signal peptide in LEKTI secretion, we constructed deletion mutants of LEKTI, expressed them in HEK 293T cells, and analyzed their secretion behavior, stability, subcellular distribution, and proteinase inhibitory function. Pro-LEKTI is processed and secreted into the medium. On the basis of partial N-terminal sequencing and immunoblotting, the cleavage products are ordered from amino- to carboxy-terminal as follows: 37, 40, and 60kDa. Inhibitors of furin lead to enhanced secretion of unprocessed LEKTI, suggesting that processing was not required for secretion. Deletion of the N-terminal signal peptide of pro-LEKTI caused altered distribution of LEKTI from endoplasmic reticulum (ER) to cytoplasm and markedly reduced its stability, consistent with its failure to become secreted into the medium. Interestingly, when we deleted the C-terminal domains, stable partial LEKTI (LD-1-6) accumulated and still retained its association with ER but was not secreted. Recombinant LD-1-6 specifically inhibited the trypsin activity. We conclude that N-terminal signal peptide is required for LEKTI import into ER and elements present in C-terminal domains may have a role in regulating LEKTI secretion.

Amino Acid Sequence↗
PubMed2002

The focal adhesion kinase amino-terminal domain localises to nuclei and intercellular junctions in HEK 293 and MDCK cells independently of tyrosine 397 and the carboxy-terminal domain.

The function and intracellular localisation of the non-catalytic NH(2)-terminal region of focal adhesion kinase (FAK) are unclear. We investigated the targetting of the FAK NH(2)-terminal domain in HEK 293 and epithelial MDCK cells. Exogenous expression of a variety of GFP-fused and epitope-tagged NH(2) terminal domain constructs either including or lacking the major Tyr 397 autophosphorylation and Src-binding site targeted to nuclei and cell-cell junctions in HEK 293 cells and co-localised at junctions with occludin, and beta1 integrin subunits at junctions. Mutation of Tyr 397 also had no effect on localisation of the NH(2)-terminal domain. In contrast, constructs encoding either the kinase or focal adhesion targeting (FAT) domains but lacking the NH(2)-terminal region failed to localise to intercellular junctions or nuclei. The NH(2)-terminal domain was not associated with beta1 integrin subunits as indicated by co-immunoprecipitation experiments, but did co-localise with cortical actin filaments. The NH(2)-terminal domain also targetted to nuclei and intercellular junctions in MDCK cells, whereas full-length FAK localised only to focal adhesions in these cells. These results indicate that the FAK NH(2)-terminal domain targets to epithelial intercellular junctions and nuclei and suggest novel functions for FAK NH(2)-terminal domain fragments independent of Y397, kinase, and FAT domains.

Actins↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗
PubMed2002

Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity.

Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.

Amino Acid Sequence↗
PubMed1997

Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Bacillus subtilis↗
PubMed1984

Tetrahymena histone H2A. Acetylation in the N-terminal sequence and phosphorylation in the C-terminal sequence.

The previous study showed that the H2A histone of the protozoan Tetrahymena pyriformis comprises equimolar amounts of two variants, H2A(1) and H2A(2), and their sequences of 137 and 132 residues, respectively, are blocked at the N-terminal and possibly partially modified in the homogeneous N-terminal and heterogeneous C-terminal regions [Fusauchi, Y. & Iwai, K. (1983) J. Biochem. 93, 1487-1497]. Now, the N-terminal blocking group was identified as alpha-N-acetyl by 1H NMR spectroscopy of the N-terminal blocked serine residue isolated by carboxypeptidase digestion of the N-terminal tryptic peptide (residues 1-5). Two epsilon-N-acetylated (16 and 14%) lysine residues positioned at 5 and 12, respectively, were found on carboxypeptidase digestion and subsequent amino acid analysis of a blocked N-terminal tryptic peptide (res. 1-8) and on aminopeptidase digestion analysis of another tryptic peptide (res. 11-17), both containing two lysine residues. The C-terminal BrCN fragment (res. 119-137) of H2A(1) containing three, two, one, and no phosphoserine, and that (res. 119-132) of H2A(2) containing two, one, and no phosphoserine were isolated by ion-exchange chromatography and determined by carboxypeptidase digestion analyses. Similar analyses of the tryptic peptides derived from these BrCN fragments showed that H2A(1) was phosphorylated at serine residues 122 (76%), 124 (15%), and 129 (25%); and H2A(2) at serine residues 122 (81%) and 128 (44%). This is the first time that C-terminal phosphorylation has been found in nucleosome-core histones.

Acetylation↗
PubMed1986

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoquinolines↗