Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi

A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.     

Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus.

The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined. It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599. The sequence of cytochrome c-552 from H. thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species. Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa. The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H. thermophilus is not high.     

Primary structure of rat insulin-like growth factor-I and its biological activities

Rat insulin-like growth factor-I (IGF-I), a serum polypeptide with growth promoting activity, was isolated from rat serum by a combination of acid/ethanol extraction, affinity chromatography, and a series of reversed phase high performance liquid chromatography, cation exchange, and reversed phase. All peptide fragments produced by chymotrypsin digestion of reduced and carboxymethylated rat IGF-I were amino acid sequenced and compared with the sequence of human IGF-I. Three out of 70 of the rat amino acid residues differed from those of human IGF-I as follows: Asp20----Pro, Ser35----Ile and Ala67----Thr. Purified rat IGF-I cross-reacted with polyclonal anti-human IGF-I antibody 75% as compared to human IGF-I, but it cross-reacted only 3% with monoclonal anti-human IGF-I antibody. Thus, it is possible to monitor the metabolic fate of human IGF-I, when injected into rats, without interference by endogenous rat IGF-I. Rat IGF-I showed 65% activity in the radioreceptor, 28.6% activity in the lipogenesis and 22.5% activity in the free fatty acid release inhibition assays as compared to human IGF-I on a protein quantity basis.     

Strain-dependent differences in susceptibility of mice to experimental Angiostrongylus costaricensis infection

Experimental Angiostrongylus costaricensis infection was carried out in inbred strains of mice (C57BL/6 BALB/c, DBA/2 and C3H/He). All strains became infected with this parasite. Marked differences in mortality and in worm burden were found among inbred strains of mice tested. A significant reduction was shown in worm length from mice compared to that from cotton rats.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.     

Procalpain I in cytoplasm is translocated onto plasma and granule membranes during platelet stimulation with thrombin and then activated on the membranes.

Thrombin stimulation of platelets resulted in changes in the subcellular localization of calpain I, with a concomitant alteration of its molecular weight as measured by immunoblotting. Calpain I in resting platelets was distributed as procalpain I, an 80 kDa form which does not exhibit the enzyme activity, and 83% of the total antigen was localized in the cytosol fraction. When platelets were stimulated with thrombin, the total content of calpain I antigen was not significantly changed as compared with that of the resting platelets, though a decrease in the cytosolic distribution of 80 kDa form (from 83% to 47% of the total antigen) was observed with concomitant appearance of the active 76 kDa and intermediate 78 kDa forms of calpain I and increase in the 80 kDa form in the granule and membrane fractions. These results indicated that calpain I was translocated from the cytosol to both the plasma and granule membranes as procalpain I and then activated on the membranes during platelet stimulation with thrombin.     

Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing.

We here report the existence of 6 additional isoforms of the NMDA receptor generated via alternative splicing by molecular analysis of cDNA clones isolated from a rat forebrain cDNA library. These isoforms possess the structures with an insertion at the extracellular amino-terminal region or deletions at two different extracellular carboxyl-terminal regions, or those formed by combinations of the above insertion and deletions. One of the deletions results in the generation of a new carboxyl-terminal sequence. All these isoforms possess the ability to induce electrophysiological responses to NMDA and respond to various antagonists selective to the NMDA receptor in the Xenopus oocyte expression system. In addition, a truncated form of the NMDA receptor also exists that contains only the extreme amino-terminal sequence of this protein molecule. These data indicate that the NMDA receptor consists of heterogeneous molecules that differ in the extracellular sequence of the amino- and carboxyl-terminal regions.     

Template-directed oligomerization of 5′-deoxy-5′-nucleosideacetic acid derivatives

5-Deoxy-5-nucleosideacetic acids II–V are isostructural analogues of nucleotides with a carboxylate group in the place of the 5-phosphate group. We have studied their oligomerization in aqueous solution using a water-soluble carbodiimide as the condensing agent in the presence or absence of an appropriate polynucleotide template. Condensation of adenylic acid analogues IIa, IIIa, and Va in the presence of polyuridylic acid were found to be the most efficient reactions. Cyclization of the activated monomers to lactones and the insolubility of the oligomers in aqueous solution were found to be obstacles to the efficient formation of long oligomers.