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Removal of extramembranous portions of the integral membrane protein Na,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaining membrane-spanning tryptic peptides. This is evidenced from comparison of the rotational mobility of native and trypsinized Na,K-ATPase using saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR spectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the protein, since the population of motionally restricted boundary lipids remains essentially the same in the native and trypsinized preparations. The ability to occlude Rb+ is also retained by the trypsinized membranes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fragment remaining when Na,K-ATPase is trypsinized in the presence of Rb+ is degraded further when the trypsinization is carried out in the presence of Na+ instead of Rb+. The rotational mobility of the tryptic fragments in the Na(+)-trypsinized membranes is lower than for the Rb(+)-trypsinized membranes, indicating rearrangement of the peptides. In addition, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the trypsinized membranes. The sequences of four membrane-spanning tryptic fragments of shark Na,K-ATPase are found to be almost identical to corresponding sequences in pig kidney Na,K-ATPase.

The amino acid sequence of human pregnancy-associated plasma protein-A (PAPP-A), a component of the circulating complex with the proform of eosinophil major basic protein (proMBP), has been determined from partial protein sequencing and from sequencing of cloned cDNA. The PAPP-A monomer contains 1547 amino acid residues, but is derived from a larger precursor of placental origin. PAPP-A contains 82 Cys residues, which are all bridged, 14 putative sites for N-glycosylation, and 7 putative sites for attachment of glycosaminoglycan groups. The C-terminal part of PAPP-A contains 5 approximately 60-residue motifs related to the short consensus repeats of complement proteins and selectins. The SCRs presently known can be grouped into three classes: complement-type, class I; selectin-type, class II; PAPP-A-type, class III. PAPP-A further contains three approximately 26-residue motifs, related to the lin-notch motifs of proteins regulating early tissue differentiation, and, in addition, a putative Zn2+ binding site similar to that found in many metalloproteinases has been identified. Apart from these features, the PAPP-A sequence is not related to other known protein sequences.

The major epsilon-lysyl-gamma-glutamyl cross-links present in the largely covalent 1:1 alpha 2-macroglobulin-(alpha 2M)-plasmin complex have been localized. Cross-linking engaged Lys607, -708, and -750 of the plasmin light chain, Lys550, -556, and -557 of the heavy chain-light chain connecting strand, Lys258 and -298 of the kringle 3 region, and Lys473 of the kringle 5 region of the plasmin heavy chain. Lys607, -708, and -750 accounted for 75% of all cross-linking, Lys550, -556, and -557 accounted for 20%, and Lys258, -298, and -473 accounted for 5%. Hence, cross-linking engaged only nine of the 41 Lys residues of plasmin, showing that, probably due to their large size, individual plasmin molecules become deposited in the large elongated binding cavity in alpha 2M in a relatively uniform way. The cross-linking of Lys residues in the heavy chain-light chain connecting strand to alpha 2M explains earlier findings that a substantial portion of the heavy chain is cross-linked to alpha 2M [Pizzo et al. (1986) Biol. Chem. Hoppe-Seyler 367, 1177-1182]. Although located in the heavy chain, Lys550, -556 and -557 should be considered part of the serine proteinase domain. That part of plasmin must be deeply buried in the alpha 2M structure in close vicinity to the four thiol esters, while most of the elongated heavy chain is protruding from the binding cavity. The pattern of cross-linked species seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that individual plasmin molecules are bound to alpha 2M through one or two cross-links. Bivalent cross-linking can take place within or between 360-kDa alpha 2M dimers, pointing to the tetrameric alpha 2M structure as the functional proteinase binding unit.

The complete primary structure of bovine beta 2-glycoprotein I was determined by a combination of cDNA and peptide sequencing. Bovine beta 2-glycoprotein I was purified from citrated plasma, and by sequencing selected peptides, the complete disulfide bridge patterns of the 11 disulfide bridges were established as well as the positions of the five asparagine-linked carbohydrate groups. Bovine beta 2-glycoprotein I comprises five mutually homologous domains or Short Consensus Repeats, each containing two disulfide bridges, except for the fifth most C-terminal domain which diverges from the Short Consensus Repeat consensus by containing an additional disulfide bridge. In the four N-terminal domains, the first and third and the second and fourth half-cystines are disulfide-linked, while in the fifth domain the first and fourth, the second and fifth, and the third and sixth half-cystines are disulfide-linked.

Human alpha 2-macroglobulin (alpha 2M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase, alpha 2M entraps the proteinase molecule in a reaction that involves large conformational changes in alpha 2M. We describe the molecular cloning of alpha 2M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human alpha 2M with a specific enzyme-linked immunosorbent assay. Human recombinant alpha 2M (r alpha 2M), secreted and purified from isolated transfected BHK cell lines, was structurally and functionally compared to alpha 2M purified from human serum. The results show that r alpha 2M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of r alpha 2M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference. Moreover, r alpha 2M-trypsin complex bound to purified human placental alpha 2M receptor with an affinity indistinguishable from that of a complex formed from serum-derived alpha 2M and trypsin.

The time course of the interactions of alpha 2-macroglobulin with trypsin and with plasmin was studied by measuring the generation of thiol groups, the concentration of alpha 2-macroglobulin subunits cleaved at the bait regions, and the change in intrinsic protein fluorescence of alpha 2-macroglobulin-enzyme reaction mixtures as functions of time. The interaction of alpha 2-macroglobulin with trypsin was found to be very fast but could be studied in the presence of benzamidine, a rather strong competitive inhibitor of trypsin. The results obtained indicate that alpha 2-macroglobulin-proteinase reactions, known to involve specific limited proteolysis of the bait regions, gross conformational changes, and cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters of native alpha 2-macroglobulin, may proceed via at least two different reaction pathways determined by the nature of or the concentration of the reacting proteinase. After initial cleavage of one bait region at high proteinase activity the next step presumably is a fast cleavage of a second bait region before any substantial rearrangements leading to generation of thiol groups and the final incorporation of the proteinase occur. At low proteinase activity no further bait region cleavages occur and only the two thiol groups of half of the alpha 2-macroglobulin molecule are generated in the final 1:1 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). Its properties and its reactions with a number of enzymes, particularly chymotrypsin, and with methylamine have been investigated. It is concluded that native PZP molecules are dimers of disulfide-bridged 180-kDa subunits and that proteinase binding results in covalent 1:1 (tetrameric)PZP-enzyme complexes. Native PZP is unstable, and storage should be avoided, but when kept unfrozen at 0 degree C most PZP preparations stay native 1-3 months. The reaction of PZP with chymotrypsin involves (i) proteolysis of bait regions, (ii) cleavage of beta-cysteinyl-gamma-glutamyl thiol ester groups, (iii) some change of the conformation and quaternary structure of PZP, and (iv) the formation of covalent 1:1 chymotrypsin-PZP(tetramer) complexes in which chymotrypsin is active but shows less activity than free chymotrypsin. The emission spectra of intrinsic fluorescence show significant differences between the PZP-chymotrypsin complex and its native components, whereas no differences are observed between methylamine-reacted PZP and native PZP. Methylamine reacts with the beta-cysteinyl-gamma-glutamyl thiol ester groups of PZP in a second-order process with k = (13.6 +/- 0.5) M-1 s-1, pH 7.6, 25 degrees C. The reaction product is PZP(dimers); no PZP(tetramers) are formed. The proteinase-binding specificity of PZP is far more restricted than that of alpha 2M. Certain chymotrypsin-like and trypsin-like enzymes are bound much less efficiently than is chymotrypsin itself.(ABSTRACT TRUNCATED AT 250 WORDS)