Biochemical characterization of serum and urinary beta 2 microglobulin in end-stage renal disease patients.

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)

Complete amino acid sequence of the sarcoplasmic calcium-binding protein (SCP-I) from crayfish (Astacus leptodactylus).

The complete amino acid sequence of the alpha chain of the dimeric sarcoplasmic Ca2+-binding protein (SCP-I = alpha 2) from crayfish (Astacus leptodactylus) has been determined by partial automatic sequencing of the peptides derived from tryptic digests of the protein after citraconylation or treatment with 1,2-cyclohexanedione. Overlapping peptides were obtained by cleavage with o-iodosobenzoic acid, or digestion with Staphylococcus aureus protease, thermolysin and pepsin. The acetylated N-terminus was identified by fast atom bombardment mass spectrometry. The monomeric protein contains 192 amino acids and has an Mr of 21,643. The sequence shows the presence of three calcium-binding sites and perhaps of two others that may be degenerated.

Fast atom bombardment mass spectra of various peptides from sarcoplasmic calcium-binding protein.

The sarcoplasmic calcium-binding protein of the crayfish Astacus leptodactylus is a calciprotein. The primary structure of this protein has been determined. Tryptic digestion of the denaturated protein followed by high-performance liquid chromatographic separation identified essentially eight peptides. The structure of these peptides has been confirmed after amino acid analysis by fast atom bombardment spectra in positive mode.

A chromatin core particle obtained by selective cleavage of histones by clostripain.

Rat liver chromatin core particles digested with clostripain yield a structurally well-defined nucleoprotein particle with an octameric core made up of fragmented histone species (designated H'2A, H'2B, H'3 and H'4, respectively) after selective loss of a sequence segment located in the N-terminal region of each core histone. Sequential Edman degradation and carboxypeptidase digestion unambiguously establish that histones H2A, H2B, H3 and H4 are selectively cleaved at the carboxyl side of Arg 11, Lys 20, Arg 26 and Arg 19 respectively and that the C-terminal sequences remain unaffected. Despite the loss of the highly basic N-terminal regions, including approximately 17% of the total amino acids, the characteristic structural organization of the nucleosome core particle appears to be fully retained in the proteolyzed core particle, as judged by physicochemical and biochemical evidence. Binding of spermidine to native and proteolyzed core particles shows that DNA accessibility differs markedly in both structures. As expected the proteolyzed particle, which has lost all the in vivo acetylation sites, is not enzymatically acetylated, in contrast to the native particle. However, proteolyzed histones act as substrates of the acetyltransferase in the absence of DNA, as a consequence of the occurrence of potential acetylation sites in the core histones thus rendered accessible. The possible role of the histone N-terminal regions on chromatin structure and function is discussed in the light of the present observations with the new core particle obtained by clostripain proteolysis.

Conformational changes induced by Mg2+ on actin monomers. An immunologic attempt to localize the affected region.

The effect of Mg2+ ions on the conformation of G-actin and in particular on the accessibility of its antigenic regions has been tested. Experiments were performed with G-actin coupled to Sepharose 4B which was, therefore, maintained in the monomeric state. The results presented her show that the 2mM MgCl2-perturbed antigenic site is located in a central region of the actin sequence.

Polymorphism in high-affinity calcium-binding proteins from crustacean sarcoplasm.

The sarcoplasmic calcium-binding proteins (SCP) from crayfish, lobster and shrimp myogen have been purified to homogeneity. These proteins exist as dimers and dissociate in the presence of sodium dodecyl sulfate or urea in subunits of 22000 molecular weight. During the last step of purification (DEAE-cellulose chromatography), SCP emerges in three peaks in the ratio of 14:1.5:1 for crayfish, of 7:2:1 for lobster and of 3:2:1 for shrimp. Gel electrophoresis and isoelectrofocusing experiments, under native and denaturing conditions, indicate that among the three SCP isotypes there are only two different polypeptide chains, alpha and beta, which appear in the form of three dimers: alpha 2, alpha beta and beta 2. The alpha and beta subunits differ slightly in polypeptide chain composition as found by amino acid analyses of the crayfish and lobster SCPs, and also by comparison of tryptic peptides for crayfish SCPs. The polymorphism observed in crustacean SCPs, which is increased by their ability to form dimers, contrasts with the situation prevailing among other invertebrate SCPs and vertebrate parvalbumins where only monomeric isotypes are found. Equilibrium binding studies show that all three SCP isotypes from both crayfish and lobster display the same metal-binding properties. They have in their dimeric form six Ca2+-binding sites: two calcium-specific sites, two Ca/Mg sites that interact with positive cooperativity and two Ca/Mg sites that interact with negative cooperativity. Interactions between the two subunits of SCP seem to result in cooperative binding of Ca2+, which in turn may control more efficiently Ca2+ fluxes in crustacean muscle.

Amino-acid sequence of an alpha-parvalbumin, pI = 4.88, from frog skeletal muscle.

The primary structure of the most basic (pI = 4.88) of the two major parvalbumins from frog skeletal muscle (Rana esculenta) has been determined by partial automatic sequencing of the protein which exhibits a free N terminus, and a study of overlapping peptides obtained by cyanogen bromide cleavage and digestion with trypsin, thermolysin and Armillaria mellea protease. This protein shows the typical structure of an alpha-parvalbumin. Comparison of the primary structure of ion-binding loops of alpha and beta-parvalbumins does not provide a clear-cut explanation of their differences in ion-binding properties.

Parvalbumins from coelacanth muscle. I. General survey.

Parvalbumins from coelacanth (Latimeria chalumnae) myogen have been isolated by gel filtration of Sephadex G-75 and DEAE-cellulose chromatography. Disc electrophoresis and cellulose acetate electrophoresis showed the homogeneity of the three first major parvalbumin peaks (pI = 5.44, pI = 4.95 and pI = 4.52). The fourth component was partially resolved into two more parvalbumins (pI = 3.78 and pI = 3.50) by preparative gel electrophoresis. Amino acid analyses and tryptic peptide maps separated the five components in two major categories. The two less acidic components differ only in the presence or absence of an N-terminal blocking group. The three more acidic components constitute the second category; in spite of this heterogeneity, they share the same amino acid sequence.

Parvalbumins from coelacanth muscle. III. Amino acid sequence of the major component.

The primary structure of the major parvalbumin (pI = 4.52) from coelacanth muscle (Latimeria chalumnae) has been determined. Sequence analysis of the tryptic peptides, in some cases obtained with beta-trypsin, accounts for the total amino acid content of the protein. Chymotryptic peptides provide appropriate sequence overlaps, to complete the localization of the tryptic peptides. Examination of the amino acid sequence of this protein shows the typical structure of a beta-parvalbumin. Its position in the dendrogram of related calcium-binding proteins corresponds to that usually accepted for crossopterygians.

Heat stability and reactivation of mare milk lysozyme.

Mare milk and aqueous solution of mare milk lysozyme were incubated for variable times between 30 C and 100 C at pH 3, 6, or 9. Lysozyme activity was stable at acid and neutral pH and labile at alkaline pH. Some of the results show the existence of a reactivation process in mare's milk and in aqueous solution. reaching 30 to 40% after incubation of the aqueous solution at 4 C for 20 days at pH 3 or 6.