Some enzymatic and physicocbemical properties of a human ubiquitin-specific isopeptidase are reported. The enzyme was purified to homogeneity from red blood cells and its specificity towards polymeric ubiquitin substrates suggests a de-ubiquitinating activity capable of cleaving 'head-to-tail' polyUb chains as well as isoamide 'branched' Ub dimers. KM values show a 10 fold preference for the cleavage of branched Ub dimers over head-totail Ub dimers. The enzymatic activity can be strongly inhibited by various peptides containing either of the cleavage site sequences found in Ub polymers, but not by unrelated peptides. The enzyme is monomeric under reducing conditions and exhibits a globular shape with an average diameter of 9 nm, an S2o,w value of 5.2 S and a molar mass of 110 kDa -+ 10%. Because the enzyme cleaves both peptide-linked and isopeptide-linked Ub moieties from substrates, we propose to name it de-ubiquitinase rather than isopeptidase.
Uridine-specific IgG and IgM antibodies, obtained by immunization of rabbits with a uridylated synthetic polypeptide, were isolated and purified by the use of a uridine-acetyl cellulose conjugate as immunosorbent. The IgM antibodies were separated from the IgG antibodies by gel filtration on Sephadex G-200. Both antibody preparations were pure by ultracentrifugation and immunoelectrophoresis criteria. The precipitating activities of IgM and IgG antibodies were similar on a weight basis, although the equivalence zone in the case of IgM antibody is broader. I n spite of its inability to precipitate the homologous antigen, the reduced alkylated IgM subunits were still able to inhibit specifically the homologous precipitation in the U-pAla-pLys -IgG antibody system. The average association constants for uridine of both IgG and IgM a.ntibodies, measured by the equilibrium dialysis technique, were low and of a similar order of magnitude.The complement fixing ability of purified IgM and IgG anti-uridine antibodies upon reaction with the homologous antigen displayed significant differences : low complement fixation activity was found in the IgM fraction either a t 4" or a t 37", while a high activity was found in the IgG fraction a t both temperatures. The IgM fraction lost all of its ability to fix complement when the macroglobulin was reduced and carboxymethylated. PCA reaction, using a highly immunospecifically purified I9 S anti-uridine antibody, was found to be negative when assayed up to an amount of 100 pg.
The assignment of disulfide bonds in human J chain and its covalent pairing with immunoglobulin M was determined under conditions which minimize disulfide bond interchange. We show that in J chain the three intradisulfide bridges are formed between Cys 12 and 100, Cys 71 and 91, and Cys 108 and 133. Previous reports [reviewed by Koshland, M. E. (1985) Annu. Rev. Immunol. 3, 425-453] have proposed that cysteines 12, 14, or 68 were linked to the penultimate cysteine 575 of two mu chain tails. In this work, we demonstrate that cysteines 14 and 68 are disulfide-bridged to mu chains. A revised, albeit putative, model of J chain folding is presented which takes into account the correct disulfide pairing and the predictive secondary structure assignment.
A cDNA clone encoding the variable region of the heavy chain of a BALB/c antibromelinized mouse red blood cell (BrMRBC) monoclonal antibody has been characterized. The nucleic acid sequence indicates that the variable region of the heavy chain is likely encoded by variable-VCP12, diversity-DSP 2 (5, 7 or 8) and joining-JH1 germ-line genes. An identical combination of V genes was observed for six other CBA/J and NZB anti-BrMRBC hybridomas. The BALB/c VCP12 nucleotide sequence is less than 80% homologous to members of the 10 known VH families. Moreover, the genomic restriction fragments detected under moderate stringency conditions with the radiolabeled cDNA probe did not correspond to those obtained with probes of the most homologous VH families (7183 and X24). The results indicate that the VCP12 gene defines a new VH family which we propose to designate VH11. The observation of 1, 2 or 3 genomic restriction fragments at the most, with mice bearing the Igh-Vd or j, Igh-Va or b or Igh-Vc haplotypes, suggests the existence of a few VCP12-related genes.
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