Distribution of the two forms of guinea-pig β2-microglobulin originally detected in urine

Two distinct forms of β₂-microglubin (β₂m) have been detected in pooled guinea-pig serum from outbred animals using ultrafiltration, gel filtration and ion exchange chromatography. These two forms probably correspond to the two β₂m forms, with different electrophoretic mobility, that have been previously identified in guinea-pig urine. In solubilized membranes from perfused guinea-pig livers only the basic form of β₂m could be found. These results may indicate that the two β₂m forms should not be ascribed to genetic polymorphism but rather are a result of post-translational modification.     

Distribution of the two forms of guinea-pig beta 2-microglobulin originally detected in urine

Two distinct forms of β₂-microglubin (β₂m) have been detected in pooled guinea-pig serum from outbred animals using ultrafiltration, gel filtration and ion exchange chromatography. These two forms probably correspond to the two β₂m forms, with different electrophoretic mobility, that have been previously identified in guinea-pig urine. In solubilized membranes from perfused guinea-pig livers only the basic form of β₂m could be found. These results may indicate that the two β₂m forms should not be ascribed to genetic polymorphism but rather are a result of post-translational modification.     

Invariant proteins associated with guinea-pig Ia antigens

Analysis of guinea-pig Ia immunoprecipitates by two-dimensional gel electrophoresis demonstrated the specific association of Ia molecules with several types of invariant proteins. These include a 33,000 mol. wt basic protein homologous to murine invariant chain (I_i), and a set of 34,000–36,000 mol. wt proteins more acidic than I_i (acidic invariant chain). Two 23,000–25,000 mol. wt nonpolymorphic proteins with pls of 6.0 and 6.5 were also observed in association with Ia, as was a basic protein of mol. wt 42,000. Pulse/chase studies using [³⁵S]methionine demonstrated that I_i, but not acidic invariant chain, was associated with newly synthesized Ia molecules. The amount of ³⁵S-I_i decreased greatly throughout the chase period. ³⁵S-acidic invariant chain was clearly present in Ia precipitates by 30 min after Ia synthesis, but was not detected 4hr after synthesis. Only acidic invariant chain was associated with mature Ia antigens bound by the lectin Ricinus communis I. Our results indicate that guinea-pig invariant proteins are differentially bound by Ia molecules during maturation of Ia - and β-chains, and suggest that acidic invariant chain could be a processed form of I_i.     

Purification and characterization of arginine carboxypeptidase produced by Porphyromonas gingivalis

Arginine carboxypeptidase was isolated from the cytoplasm of Porphyromonas gingivalis 381 and purified by DEAE-Sephacel column chromatography, followed by high-performance liquid chromatography on DEAE-5PW and TSK G2000SW(XL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of three major bands at 42, 33, and 32 kDa with identical N-terminal sequences. By Western blotting analysis and immunoelectron microscopy, the arginine carboxypeptidase was found to be widely distributed in the cytoplasm and on the surface of the outer membrane. The open reading frame corresponding to the N-terminal amino acids of the arginine carboxypeptidase was detected by a search of the sequence of the P. gingivalis W83 genome. This sequence showed homology with mammalian carboxypeptidases (M, N, and E/H) and included a zinc-binding region signature, suggesting that the enzyme is a member of the zinc carboxypeptidase family. The purified enzyme was inhibited by EGTA, o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and some metal ions, such as Cu(2+), Zn(2+), and Cd(2+). On the other hand, Co(2+) activated the enzyme. The enzyme released arginine and/or lysine from biologically active peptides containing these amino acids at the C terminus but did not cleave substrates when proline was present at the penultimate position. These results indicate that the arginine carboxypeptidase produced by P. gingivalis is an exo type of metallocarboxypeptidase. This enzyme may function to release arginine in collaboration with an arginine aminopeptidase, e.g., Arg-gingipain, to obtain specific amino acids from host tissues during the growth of P. gingivalis.     

Rheumatoid factors from patients with rheumatoid arthritis react with Des-Lys58-beta 2m, modified beta 2-microglobulin.

Ten polyclonal IgM rheumatoid factor (RF) preparations, affinity-purified from IgG columns, from patients with rheumatoid arthritis were studied for their ELISA reactivity with native beta 2m in parallel with Lys58-cleaved beta 2m and Des-Lys58-beta 2m, the latter representing cleavage products of the native molecule present in some pathologic human sera. Most RF showed positive reactions with the native form of beta 2m but reduced reactivity for the cleaved forms of beta 2m. Reactions between cleaved beta 2m and RF, in solution, were demonstrated by inhibition of RF binding to native beta 2m by preincubation with a range of concentrations of Des-Lys58-beta 2m. By contrast, eight of nine murine MoAbs to human beta 2m showed approximately equivalent binding to native beta 2m, Lys58-cleaved beta 2m, and Des-Lys58-beta 2m. Reactions between individual human RF and the altered forms of beta 2m (Lys58-cleaved beta 2m and Des-Lys58-beta 2m) appeared to parallel the previously determined beta 2m single amino acid specificities, in that RF showing strong reactivity with Lysine 58 also showed a significant diminished reactivity with the Des-Lys58-beta 2m lacking the critical lysine residue. The present studies demonstrate that while human RF react with Lys58-cleaved beta 2m or Des-Lys58-beta 2m, preferential reactivity is observed for native unaltered beta 2m.     

The guinea-pig I-region. III. Distribution of Ia antigens in inbred and partially inbred strains

We have used a combined serologic and structural approach to study the distribution of I-region associated (Ia) antigens in nine strains of inbred and partially inbred guinea-pigs. All of the inbred strains studied with the exception of strain 2 animals were found to share one or more I-subregions with inbred strain 13 animals. The BIOAD, R9, OM3, and BIOAC strains have the same I-region as strain 13 animals; the B/Lac strain has two subregions in common with strain 13, while the BIOB strain has a single subregion in common with strain 13. The availability of a number of different guinea-pig strains with well-characterized major histocompatibility complexes should facilitate the continuing use of this species in studies of immunogenetics, transplantation, and tumour immunology.     

Charge heterogeneity of beta 2-microglobulin in lymphoid cells

Extracts of blood lymphocytes, polymorphonuclear neutrophils and B, T or monocytic cell lines were analyzed by two-dimensional gel electrophoresis and immunoradiometric assay after electro-transfer to nitrocellulose sheets with radiolabelled polyclonal or monoclonal antibodies specific for beta 2-microglobulin. Four different forms of the molecule were identified with an apparent Mr of 12,000 and pI values of 5.7, 5.3 and lower. Lymphocyte activation by phytohemagglutinin and concanavalin A, or incubation with recombinant alpha 2b interferon, resulted in an increased beta 2-microglobulin cell content and release of the protein in supernatants with a predominant elevation of the more acidic minor forms. Recombinant interleukin-2 and recombinant gamma interferon increased the expression of the molecule without significant shift in the relative proportion of beta 2-microglobulin forms. Tumor necrosis factor alpha did not increase cell beta 2-microglobulin (beta 2-m) content and release and did not alter the relative distribution of the different forms of the molecule. Several mechanisms may be considered for the generation of beta 2-m microheterogeneity, including intracytoplasmic post-translational modifications such as proteolysis or modification of the amide groups of internal amino acids.     

A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein

The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic amino acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids.     

Binding of β2-Microglobulin by Heterologous Sera

Purified human, rat or guinea-pig beta 2-microglobulin (beta 2m) was mixed with sera from guinea-pig, rat, mouse, rabbit, horse, goat, cow, rhesus monkey or man. The mixtures were incubated at 37 degrees C for various lengths of time. When the sera were separated by gel-chromatography on Sephadex G-200, beta 2m was traced not only in 'free' form but also in fractions with higher molecular weights. Evidence is presented suggesting that heterologous beta 2m binds to beta 2m-containing molecules in sera by exchange with the homologous counterpart.     

The major histocompatibility complex of the guinea-pig. II. Relationship between mixed leucocyte reactivity and serologically-defined phenotypes of the GPLA B locus and I region.

The relationship between the mixed leucocyte reaction (MLR) and the serologically-defined antigens controlled by genes in the guinea-pig B locus (equivalent to the murine D or K region) and I region was investigated in various inbred and partially inbred strains and in guinea-pig families homozygous for their GPLA alleles. No MLR reactions could be detected among guinea-pig families of a closed colony which had been bred to homozygosity for their GPLA antigens. By contrast, animals which differed in their I region showed strong MLR reactivity. Animals with identical I regions as judged by four serologically-defined specificities only, but differ with respect to B locus determinants, yield appreciably lower MLR responses. It appears that in the guinea-pig, as in other mammalian species, the antigenic systems responsible for MLR reactivity are controlled primarily by genes identical with or closely linked to the I region of the major histocompatibility complex.     

Isolation of a low-molecular-weight antibacterial system from human amniotic fluid.

A low-molecular-weight antibacterial system has been isolated from human amniotic fluid. The bacterial inhibitor requires the metal cation zinc and a peptide with a molecular weight of 630. The peptide component was purified using ultrafiltration, gel filtration, and ion-exchange chromatography. It can be inactivated by digestion with carboxypeptidase. The amino acid composition of the peptide is: 3 glutamine-glutamic acid, 2 glycine, and 1 lysine. Removal of zinc from the peptide has been shown to remove bacterial inhibitory activity.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9-7.0), octameric (pI 7.0-7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8-6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

Molecular heterogeneity of amyloid beta2-microglobulin and modification with advanced glycation end products

By using liquid chromatography-electrospray ionization mass spectrometry, Western blotting and N-terminal amino acid sequence analysis, we characterized the molecular heterogeneity and advanced glycation end product (AGE) modification of beta2-microglobulin (beta2m) extracted from the amyloid tissue of a hemodialysis patient. Amyloid beta2m was composed of full-length beta2m, truncated beta2m and dimer beta2m. Truncated beta2m and dimer beta2m were modified with AGEs such as imidazolone and N(e)-(carboxymethyl)lysine, and showed fluorescence characteristic of AGE. Truncated beta2m species were formed by cleavage between amino acid residues of Pro6/Ile7, Gln/Val9 and Val9/Tyr10. Heterogeneous dimer beta2m species showed the molecular masses of 22,591 and 22 675, which resulted from cross-linking between truncated beta2m.     

The transamination of glutamate and aspartate during absorption in vitro by small intestine of chicken, guinea-pig and rat

1. Procedures are described for direct measurement of the extent and rate of transamination of glutamate and aspartate over periods of up to 90 min, during absorption in vitro by the small intestine of chicken, guinea-pig, and rat.2. During absorption of dicarboxylic amino acids by rat small intestinal segments circulated through the lumen in vitro, alanine contributed up to 85% of the amino acids appearing in the fluid secreted at the serosal surface. In guinea-pig and chicken intestine, the proportion of alanine in the secreted amino acids did not exceed 60%.3. For the different species studied, a relationship was found between the extent to which the dicarboxylic amino acids were transaminated to alanine and the total amount of GPT found in other studies to be present in the intestinal mucosa. In both rat and guinea-pig small intestine, the proportion of alanine in the total amino acids appearing at the serosal surface was similar in the jejunum and ileum. The rate of appearance of alanine in serosal fluid was greater in the ileum than in the jejunum of the rat.4. Reasons are given for supposing that for all the species studied there is a limit to the capacity of the small intestinal mucosa to subject free dicarboxylic amino acids to transamination. It is concluded, however, that it is unlikely that this capacity will be exceeded under in vivo conditions.     

人源性羧肽酶 A突变体基因的构建及表达

目的从人正常肺组织中提取羧肽酶A基因并进行定点突变。方法从人正常肺组织中提取RNA,并进行逆转录得到人羧肽酶A(hCPA)。以计算机模拟hCPA的结构,寻找合适的突变位点。用PCR重叠延伸法,对羧肽酶A进行定点突变,并在大肠杆菌中表达。结果序列分析表明,所获hCPA的核苷酸序列为1251bp,编码417个氨基酸。hCPA突变体(mhCPA)基因的1126位核苷酸由G变为A,其余核苷酸序列均未发生变化,相应的此突变体蛋白质的376位核苷酸残基由丙氨酸突变为苏氨酸。将mhCPA基因在E.coli中诱导表达3h后,表达量高达30％左右。结论成功地构建并表达了mhCPA基因,为hCPA在肿瘤的“抗体导向酶前药疗法”(ADEPT)中的应用奠定了基础     

GPLA antigens are not expressed on guinea-pig epididymal spermatozoa

The expression of guinea-pig major histocompatibility antigens (class I and II) has been investigated on guinea-pig epididymal spermatozoa. Specific alloantisera (anti-B1, anti-B3, anti-Ia2,4 and anti-Ia1,3,7) were obtained by cross-immunization of strains 2, 13 and BIO-AD animals with relevant spleen cell membranes. These sera were tested on spermatozoa from the same three strains by a protein A rosetting assay and by an indirect immunofluorescence test. The results obtained showed non-strain specific reactions of all the alloantisera tested on epididymal spermatozoa of the three strains. These non-strain specific reactions were absorbed by spermatozoa of any strain but they were not absorbed by the splenic cells of the same animals. On the other hand, the alloantisera specific reactivity on peripheral blood cells was not diminished after incubation of the sera with spermatozoa. Furthermore anti-B1 and anti-B3 antibodies eluted from guinea-pig platelets did not react with any spermatozoa but reacted with the relevant peripheral blood cells. These results indicate that the studied guinea-pig sera contained two types of antibodies: anti-MHC antibodies able to react with relevant blood cells but not with spermatozoa and sperm specific antibodies (also observed in untreated and DNP-BGG immunized guinea-pig sera) reacting with all spermatozoa but not with blood cells. They are not compatible with the expression of MHC antigens at the surface of guinea-pig epididymal spermatozoa.     

Thialysine-resistant mutants and uptake of lysine in Schizosaccharomyces pombe

Summary Mutants defective in lysine transport were isolated and characterized. After UV-mutagenesis colonies resistant to thialysine, a toxic analogue of lysine, were isolated and L-lysine uptake into the mutant strains was analyzed. Among the thialysine-resistant strains a group of mutants was found, where the half-saturation constant, K_T, of the high-affinity transport system for lysine was higher than in the wild-type, the high-affinity transport system for basic amino acids being specifically affected. This was confirmed by a complementation test in which all the thialysine-resistant strains with a higher K_T for lysine uptake belonged to one complementation group. Kinetic and genetic analysis showed that our mutants were identical with can1-1 mutants, showing that a single high-affinity system for the transport of basic amino acids exists in S. pombe.I.B.M.C., C.N.R.S., 15, rue René Descartes, F-67000 Strasbourg, France     

Isoelectric focusing of human and guinea-pig C2: polymorphism of guinea-pig C2

The isoelectric point (pI) of human and guinea-pig C2 was determined by electrofocusing. The results showed that human C2 and C2 of some guinea-pigs had a pI of approximately pH 5.5–5.6; in some guinea-pig sera C2 activity was resolved into two fractions, one with a pI of about 5.2 and the other of about 5.5. The data suggest the possibility that guinea-pig C2 may exist in at least two allotypic forms.     

Purification and characterization of mouse beta-2 microglobulin: allelic variants from two different strains

Beta-2 microglobulin (beta 2M) is a 12,000 dalton protein associated with membrane-bound cell surface antigens. Variants of beta 2M, beta 2MA and beta 2MB, were first detected by Michaelson et al. (Immunogenetics 11, 93-95, 1980). An improved method was used to purify beta 2MA and beta 2MB from BALB/c and C57BL/6 mouse livers, respectively. Reproducible yields of 10% were obtained. The purifications were accomplished by a 3 M sodium thiocyanate (NaSCN) extraction of a crude membrane fraction, an acid precipitation step, gel filtration on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose and CM-cellulose in that order. The elution profile of beta 2MA and beta 2MB on the ion-exchange columns was found to be different, indicating the presence of structural changes. beta 2MA was found to be more acidic (pI = 7.35) than beta 2MB (pI = 7.68) by isoelectric focusing in gels. Complete sequence analysis of beta 2MA and partial sequence analysis of beta 2MB (61 of 99 residues) were performed by automated Edman degradation of the intact chain and of the overlapping peptides obtained by: (a) tryptic cleavage at arginines after acetimidation of lysine side chains, (b) BNPS-skatole cleavage at tryptophan residues and (c) hydroxylamine cleavage at asparagine-glycine linkages. A comparison of the primary structure of beta 2MA to the partial amino acid sequence obtained for beta 2MB revealed a single amino acid substitution (aspartic acid for alanine at position 85) that accounts for the differences in biochemical properties observed.     

The effect of amino acids on the intestinal absorption of immunoglobulins in the neonatal rat

An in vitro preparation of 10-day-old rat intestine was used to examine the absorption of a number of amino acids and immunoglobulins. Evidence was obtained for the active absorption of alanine, leucine, methionine, histidine and lysine, but not for aspartic acid. A selective absorption of the homologous molecule was found in experiments where ¹³¹I-labelled rat and bovine IgG were presented to the ileum in 10-minute incubations. The greater uptake of rat IgG was unrelated to the relative rates of catabolism of the two molecules.

Although the uptake of rat IgG was unaffected by 100 mM concentrations of neutral and acidic amino acids, the basic amino acids arginine and lysine significantly stimulated uptake.