Tryptic digestion of brain spectrin generates a number of fragments from α and β subunits; when these fragments are incubated with F‐actin or neurofilament light subunit, four of them with molecular masses below 30 kDa sediment with the cytoskeleton structures. A selective purification of these fragments by ammonium sulfate fractionation and butyl‐Sepharose chromatography has been achieved. Two fragments with molecular masses of 28 and 23 kDa bind to F‐actin. Native brain spectrin causes half‐maximal inhibition of the association at a concentration of 3 μM. Protein sequencing indicates that the actin‐binding domain is contained in the β subunit, in a stretch of amino acids at the N terminus from Ala43 (28‐kDa fragment) or from Met104 (23‐kDa fragment) and terminate probably at the C‐terminal Lys288 or Lys284. Amino acids are numbered by reference to the sequence of the Drosophila β subunit. The 28‐kDa fragment also binds to the low‐molecular‐mass subunit of neurofilaments; brain spectrin heterodimer disrupts this binding. Hence, spectrin binds to F‐actin and to neurofilaments via a common binding domain.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIsolation and characterization of a rat skin parvalbumin-like calcium-binding proteinMurielle L. Rinaldi, Jacques Haiech, Jana Pavlovitch, Marthe Rizk, Conception Ferraz, Jean Derancourt, and Jacques G. DemailleCite this: Biochemistry 1982, 21, 19, 4805–4810Publication Date (Print):September 14, 1982Publication History Published online1 May 2002Published inissue 14 September 1982https://pubs.acs.org/doi/10.1021/bi00262a044https://doi.org/10.1021/bi00262a044research-articleACS PublicationsRequest reuse permissionsArticle Views48Altmetric-Citations44LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAbolition of ATPase activities of skeletal myosin subfragment 1 by a new selective proteolytic cleavage within the 50-kilodalton heavy chain segmentPatrick Chaussepied, Dominique Mornet, Etienne Audemard, Jean Derancourt, and Ridha KassabCite this: Biochemistry 1986, 25, 5, 1134–1140Publication Date (Print):March 11, 1986Publication History Published online1 May 2002Published inissue 11 March 1986https://pubs.acs.org/doi/10.1021/bi00353a028https://doi.org/10.1021/bi00353a028research-articleACS PublicationsRequest reuse permissionsArticle Views30Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
F‐actin specifically substituted with the photocross‐linker, p ‐azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment‐1 (S1) and photolysis at 365 nm, it was readily cross‐linked to the S1 heavy chain with a yield of about 13–25%, generating four major actin–heavy‐chain adducts with molecular masses in the range 165–240 kDa. The elevated Mg 2+ ‐ATPase of the covalent complexes displayed a turnover rate of 33±8 s −1 which is similar to the values reported earlier for other acto‐S1 conjugates. The cross‐linking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross‐linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50‐kDa region, whereas that attached to Arg28 mediated the selective cross‐linking of the COOH‐terminal 22–21‐kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50‐kDa and 20‐kDa fragments. The rapid photoactivation and cross‐linking to S1 of the substituted F‐actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain‐1 of actin during the ATPase cycle.
Since the identification of beta 2 microglobulin (beta 2-M) in haemodialysis-associated amyloidosis, the biochemical characterization of the different forms of beta 2-M has been sought by several groups. New beta 2-M isoforms (pI 5.1 and lower) have been identified in amyloid deposits, and it has been suggested that they are of pathogenetic importance. The finding of N-terminal proteolysed beta 2-M in amyloid deposits prompted the hypothesis that proteolysis would render beta 2-M more amyloidogenic. Finally, a 'novel beta 2-M' (pI 5.2) with a single amino acid replacement (Asn by Asp at position 17) has been reported as possibly specific for patients with dialysis associated amyloidosis, and consequently proposed as 'the amyloidogenic' form. We purified beta 2-M from serum of a newly haemodialysed patient and from urine of a transplanted patient in the early recovery period. Both patients were clinically amyloid free. Three pure isoforms were obtained from serum (pI 5.7, 5.3, and 5.1) and only two from urine (5.7 and 5.3). Further purification of each isoform was obtained by HPLC in a C4 column. Sequence analysis showed that all isoforms had an intact N-terminus. Tryptic digestion of the serum isoforms was performed after alkylation with iodoacetic acid and the peptides were isolated by HPLC in a C18 column. The 5.3 and 5.1 isoforms had identical peptide patterns with the appearance of an early peak missing in the 5.7 form. The sequence of this peptide showed a replacement of the D 42 (Asp 42) by N (Asn) after K41 (Lys 41).(ABSTRACT TRUNCATED AT 250 WORDS)
The existence of the two connector segments linking the tryptic 50 kDA fragment of skeletal S1 heavy chain to the adjacent 27 kDa and 20 kDa peptides was ascertained by digestion of S1 with staphylococcal protease which was found to act specifically at these particular regions. Three new peptides of M r 28 000, 48 000 and 22 000 were produced and the novel S1 derivative formed had an intact actin‐activated ATPase activity. Amino acid sequence analyses indicated that the 48 kDa and 22 kDa peptides overlap the two connector elements.
A cytochrome P-450 involved in the metabolism of cyclosporin A (CsA) was isolated and purified to electrophoretic homogeneity from human liver microsomes of renal transplant donors. This cytochrome, designated P-450(CsA), exhibited a type I binding spectrum in the presence of CsA with a Ks(app) of 25 microM, a molecular weight of 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a maximal absorbance at 449 nm when reduced in the presence of carbon monoxide. The N-terminal sequence of P-450(CsA), determined by Edman degradation reaction, was 63% homologous with that of the rabbit liver CsA oxidase P-450 3c and 100% homologous with that of the human liver isozyme P-450(HLp/NF), recently identified as the human nifedipine (NF) oxidase. Polyclonal and monoclonal antibodies directed against P-450 3c and P-450(HLp/NF), respectively, recognized native microsomal and highly purified P450(CsA). As observed in the rabbit, human liver microsomes were shown to generate mono- and dihydroxy, as well as dihydroxy and/or monohydroxy N-demethylated, derivatives of CsA. Production of these metabolites was shown to be specifically inhibited by anti-P-450 3c polyclonal antibodies. CsA oxidase, NF oxidase, and erythromycin demethylase were shown to be closely correlated with the level of P-450(CsA) determined from Western blot or enzyme-linked immunosorbent assay. Moreover, these monoxygenase activities and the hepatic level of P-450(CsA) were simultaneously increased in the liver of patients treated for 4 days with 600 mg of rifampicin per day. Finally, NF was shown to be a competitive inhibitor of CsA oxidation and vice versa. We conclude that P-450(CsA) is responsible for most (80%) of CsA oxidase activity in human liver, is encoded by gene P450IIIA3, as is NF oxidase, or a very closely related gene, and is strongly inducible by rifampicin pretreatment.
Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.
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