Journal of applied biochemistry最新文献

A tetradecapeptide, H-Arg-Lys-Asp-Val-Tyr-Val-Glu-Leu-Tyr-Leu-Gln-Ser-Leu-Thr-OH, corresponding to amino acids 32 to 45 of thymopoietin II and its six analogs by replacing the amino acid residue in position 37, was prepared. These peptides were synthesized using conventional synthesis in solution and were tested for their effect on impaired T-cell transformation by phytohemagglutinin (PHA) in the common variable immunodeficiency. The tetradecapeptide had increasing activity on the T-cell transformation by PHA. Among the tetradecapeptide analogs, several analogs in which Val37 was replaced by Leu or Phe exhibited potent activity which was more than that of the parent peptide fragment. On the other hand, replacement of Val37 by Pro, beta Ala, or sarcosine had no effect at concentrations as high as 3.5 X 10(-4) M. One analog whose Val37 was replaced by Gly showed activity one-third that of the parent peptide fragment. On the basis of these results, the structure-activity relationship for the tetradecapeptide is discussed.

To understand the in vivo actions of angiotensin-converting enzyme (ACE) inhibitors, a prolonged study was performed in rabbits over a half year, using one of such inhibitors, foroxymithine. During the initial 2 months of the inhibitor administration, the serum level of ACE was suppressed. Thereafter, probably triggered by the consequent sharp rise in the plasma renin activity (PRA) level, the ACE level regained its initial value. Thus the close correlation between the levels of PRA and ACE seen in the control animal was entirely broken by this inhibitor. A multivariate study indicated that the inhibitor drastically changed the normal networks of peptide metabolism in vivo. These results are compatible with the notion that the ACE inhibitor blocks the regulatory mechanisms of the renin-angiotensin system in vivo.

A modified peptide mapping technique is described that allows the survey of the primary structure of proteolytic fragments of particular newly translated proteins in reticulocyte lysate. The technique is demonstrated with rat liver cytochrome b5.

The preparation of adsorbents for DNA antibodies is described. The degree of immobilization of native DNA on Sepharoses activated with epichlorohydrin or bisoxirane was investigated as a function of pH, temperature, time, concentration of DNA, and oxirane content in the supports. The maximum amount of DNA bound was obtained after 8 h at 40-50 degrees C at pH 11-11.5. The amount bound was increased by raising either the concentration of DNA or the oxirane content of the supports, and could reach 300 mg/g dry support. The immobilized DNA was applied to the adsorption of DNA antibodies using either commercial human serum with anti-native DNA activity or the sera of patients with systemic lupus erythematosus. The amount of antibody adsorbed depended on the amount of DNA. The thermal stability of the immobilized DNA was also examined. After heating at 80 degrees C, the leakage of DNA was slight and the adsorption of antibodies was not affected.

Previous studies indicated the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases. In the present study, we examined the activities of such enzymes in various organs of the hybrids of the New Zealand Black and New Zealand White mouse (NZB/W mouse) as a laboratory model of human systemic lupus erythematosus. Of the 18 enzymatic activities tested, the activities of the post-proline-cleaving enzyme showed a particular behavior in the spleen of NZB/W mouse. The enzymatic activity progressively increased with age in contrast to the reverse tendency in the control. This phenomenon was not found in any organs other than the spleen. The activity of this enzyme showed a high level of correlation to that of proline-iminopeptidase in most organs tested from control animals. However, these correlations were almost completely absent in the spleen of NZB/W mice. This may suggest an important pathogenetic role for the post-proline-cleaving enzyme in immunological disturbances in this model animal.

Studies were carried out to assess the prospects of adapting an enzyme administration procedure developed with rat liver gulonolactone oxidase to other enzymes of therapeutic interest. The enzyme is administered intraperitoneally as the glutaraldehyde-reacted immunoprecipitate. A gulonolactone oxidase from a different source, chicken kidney, also shows catalytic capability following administration. This finding suggests that other enzymes modified by this procedure might also act in vivo. Four out of five enzymes tested (asparaginase, serum cholinesterase, rat and chicken gulonolactone oxidases) have significant catalytic activity and relatively minor changes in affinity for substrate after the modification, and only one (histidase) is inactivated by the modification. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these enzymes indicates that they consist largely of enzyme and immunoglobulin G. All five of these modified enzymes are not toxic even with repetitive administrations whereas unmodified asparaginase is allergenic to a majority of guinea pigs tested. The modification described is very simple and rapid and is, therefore, a practical means of preparing certain enzymes for therapeutic administration.

DNA was immobilized covalently to Sepharose by several methods using epichlorohydrin, cyanogen bromide, carbodiimide, hydroxysuccinimide, carbonyldiimidazole, trichlorotriazine, and diazonium salt. These immobilizing methods were compared from the standpoint of the preparation of immunosorbent for anti-DNA antibodies. Among these methods, that involving epichlorohydrin was the most suitable because of large coupling capacity, stability of bound DNA, and nonadsorption of anti-DNA by the support itself.

Methods for the specific conjugation of both polyclonal antibodies and monoclonal antibodies via their carbohydrate moieties are described. Mild oxidation of the immunoglobulin with sodium periodate produces reactive aldehydes on the carbohydrate moieties. Subsequent reaction with hydrazide derivatives of biotin, fluorescent dyes, or enzymes produces stable antibody conjugates which retain full immunological activity. In addition, immunoaffinity supports can be prepared in the same manner using solid supports containing a hydrazide function.

A generally applicable approach to the preparative isolation of amphiphilic membrane proteins that follow the Triton X-114 phase during a temperature-dependent phase separation is described. The phase separations were performed direct on whole blood and a 650-fold purification of human erythrocyte membrane acetylcholinesterase (AchE) was obtained. Thus, 0.2 mg enzyme was isolated per 1 liter of blood, with a specific activity of 13 IU/mg, the major contaminants being glycophorin and hemoglobin. The protein material was isolated from the detergent phase by Cu2+ chelate chromatography. This material was used to raise monoclonal anti-AchE antibodies which, when applied to immunosorbent chromatography of washed Triton X-100-lysed erythrocytes in one step, allowed a 246,000-fold purification of AchE with a yield of 88% and a specific activity of 3800 IU/mg.

Calmodulin binds quantitatively to phenyl-Sepharose through its Ca2+-induced hydrophobic binding region. Troponin C and S-100 protein, as well as several other proteins present in rat tissues, also bind to phenyl-Sepharose in a Ca2+-dependent manner. While the Ca2+-dependent binding of calmodulin to phenyl-Sepharose is not altered appreciably by monovalent cations, they do appear to compete for Ca2+ binding to most of the other proteins, including S-100 protein, which exhibit Ca2+-induced interaction with phenyl-Sepharose. The selective elution of these proteins from the phenyl-Sepharose column can be achieved with a 0.5 M concentration of monovalent cations such as K+, Na+, and NH4+ in the presence of a low (100 microM) Ca2+ concentration. Calmodulin-binding proteins associated with calmodulin in crude cell extracts can prevent the interaction of calmodulin with the phenyl-Sepharose, resulting in low recoveries of calmodulin from these tissues. The majority of these interfering proteins are heat labile so that heat treatment (boiling) of the cell extract for a limited time (5 min) negates any binding of these proteins to calmodulin and allows the quantitative recovery of calmodulin by hydrophobic interaction chromatography. This procedure allows the rapid and quantitative recovery of highly purified calmodulin from both cytosolic and Triton X-100-solubilized particulate fractions prepared from various rat tissues. Calmodulin isolated in this manner can be accurately and reliably quantitated by direct protein determination with Coomassie brilliant blue dye or fluorescamine or by the cyclic nucleotide phosphodiesterase stimulation assay.