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.

Reciprocal coupling between troponin C and myosin crossbridge attachment.

The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.

Structure of a gene for a lysosomal membrane glycoprotein (LEP100). Housekeeping gene with unexpected exon organization.

Members of a recently described family of glycoproteins constitute the major protein components of the lysosomal membrane. Changes in glycosylation patterns, redistribution to the cell surface, and increased levels of expression of these proteins are associated, in at least some cases, with cell differentiation, transformation, and metastasis. To understand further the regulation of expression of these proteins and their relationships to each other, we have isolated and characterized the gene for one of these proteins, LEP100, from chicken. Two overlapping clones were isolated which contain the nine exons and eight introns of the 17-kilobase gene for LEP 100. The 5'-flanking region of the gene contains CAAT and TATAAA sequences, but these do not appear to be used as promoter elements. Further downstream are three CCAAT boxes, with no corresponding TATA boxes, which represent putative promoter elements. Multiple CCAAT boxes, the apparent lack of a TATA box, and the GC-rich composition of the 5' region support the classification of the LEP100 gene as a TATA box-lacking housekeeping gene. As further evidence of the housekeeping nature of the LEP100 gene, Northern blots of RNA from several adult and embryonic tissues (skeletal muscle, kidney, liver, heart, gizzard, and brain) revealed a single message for LEP100 of the same size (about 3 kilobases) in each tissue. The gene's introns range in size from 104 to 7200 base pairs. Exons do not represent the four disulfide-bonded loops of the protein, but instead each cysteine of each disulfide-linked pair is encoded by a separate exon. The existence of the same sized mRNA in all tissues indicates that no alternate splicing occurs. The exon organization of the LEP100 gene suggests that it may have evolved from a primordial cysteine-containing exon by gene duplication events. It is likely that the genes of the other members of this family diverged from the same ancestral gene and have a gene organization similar to that of LEP100.

The effect of [Mg2+] on the Ca2+ dependence of ATPase and tension development of fast skeletal muscle. The role of the Ca2+-specific sites of troponin C.

Conflicting reports have appeared concerning the effect of [Mg2+] on muscle activity. Several groups have found that increasing [Mg2+] produces a right-ward shift of the pCa-tension curve, while others have found no effect of [Mg2+] on myofibrillar ATPase activity. The present study is a careful evaluation of the effect of [Mg2+] on myofibrillar ATPase, skinned fiber tension development, TnCDANZ (troponin C (TnC)-labeled with 5-dimethylaminonaphthalene-1-sulfonyl aziridine) fluorescence, and simultaneous TnCDANZ fluorescence and tension development in the same fiber. A small effect of [Mg2+] on both ATPase and tension development was found with an apparent association constant of about 2 X 10(2) M-1. The Ca2+ dependence of TnCDANZ fluorescence was similarly effected by [Mg2+], either alone or when incorporated into TnC-depleted skinned fibers (K'Mg approximately equal to 2-3 X 10(2) M-1), suggesting that the effect of [Mg2+] on activity is due to an effect of [Mg2+] on Ca2+ binding to the Ca2+-specific sites of TnC. It is not yet clear whether this effect of [Mg2+] is through direct competition at the binding sites or through indirect effects. In either case, the calculated effect of physiological [Mg2+] is so small that the regulatory sites of TnC can still be considered "Ca2+-specific." In addition, a slightly greater effect of [Mg2+] on tension development (K'Mg = 4.62 X 10(2) M-1) was observed only for very low levels of [Mg2+], which might suggest an additional effect of Mg2+ on tension development which is saturated by millimolar Mg2+.

Identification of Acan125 as a myosin-I-binding protein present with myosin-I on cellular organelles of Acanthamoeba.

We have discovered the first protein to bind to a non-filamentous myosin, aside from actin. This protein, Acan125, is a 125-kDa protein from Acanthamoeba that associates with the SH3 domain of Acanthamoeba myosin-IC and not the SH3 domain of human fodrin. Antibodies raised against Acan125 recognize a single protein of 125 kDa from a whole cell lysate of Acanthamoeba; antibodies to myosin-I (M1.7 and M1.8) do not recognize Acan125 on the same blot. Double labeling of Acanthamoeba show Acan125 and myosin-I to be present on the same intracellular organelle, most likely amoebastomes. Immunoprecipitation with either anti-myosin-I or anti-Acan125 antibodies coprecipitates both Acan125 and myosin-I from a lysate of Acanthamoeba, demonstrating that Acan125 interacts with native myosin-I.

Isolation of two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum.

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.

Isolation and sequence of a cDNA clone for rabbit fast skeletal muscle troponin C. Homology with calmodulin and parvalbumin.

The binding of Ca2+ to troponin C (TnC) regulates skeletal muscle contraction. We have isolated a full-length cDNA clone for fast skeletal muscle TnC from a neonatal rabbit skeletal muscle library and determined its nucleic acid sequence. The amino acid sequence deduced from this clone matches the previously reported amino acid sequence (Collins, J. H., Greaser, M. L., Potter, J. D., and Horn, M. J. (1977) J. Biol. Chem. 252, 6356-6362) except at the amino terminus. According to the nucleotide sequence, the first 2 residues of TnC are threonine-aspartic acid, which is the reverse of the order reported previously. The isolation of the adult form of TnC from a neonatal library suggests that there may be no developmental isoforms of fast TnC. The protein coding region of the fast TnC clone has 67% homology with the reported nucleotide sequence for chicken slow TnC (Putkey, J. A., Carroll, S. L., and Means, A. R. (1987) Mol. Cell. Biol. 7, 549-1553). The homologies between the nucleotide sequences of TnC, calmodulin, and parvalbumin provide evidence that all three proteins were derived from a common precursor molecule which had four Ca2+-binding sites.