Identification of actin and HSP 70 as cyclosporin A binding proteins by photoaffinity labeling and fluorescence displacement assays.

A novel family of cyclosporin A (CsA) binding proteins was identified by using the biologically active, radioiodinated photoaffinity probe [D-Lys-N epsilon-(4-azido-3-[125I]iodophenyl)propionyl)]8-CsA. In addition to cyclophilin, proteins with molecular masses of 43 kDa and approximately 50-55 kDa were labeled in Jurkat extracts and bovine calf thymus. Sequence analysis of the 43-kDa protein purified from calf thymus and subsequent Western analysis of CsA affinity-purified material from Jurkat extracts identified the 43-kDa component as actin. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA, a fluorescent analogue of CsA, was prepared and used to measure the binding constants of cyclosporin derivatives to actin by means of a new fluorescence displacement assay. [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA and [N delta-t-butoxycarbonyl diaminobutyryl)]8-CsA bind to bovine actin at physiologically relevant concentrations, with dissociation constants of 60 +/- 33 and 570 +/- 380 nM, respectively. Because the ATPase fragment of heat shock cognate 70 (HSC 70) is structurally related to actin, the yeast homologue SSA1 was tested and found to be radiolabeled by the cyclosporin A photoaffinity reagent. The binding constant for [D-Lys-N epsilon-(5-dimethylamino-1-naphthalenesulfonyl)]8-CsA to SSA1 was determined and is 53 +/- 48 nM. These results indicate that actin and the 70-kDa heat shock protein family contain a structurally related domain for binding of cyclosporin A-related peptides.     

The Chemical Fractionation of Rabbit and Swine Thymus

A chemical fractionation procedure, previously found applicable to bovine thymus and bovine and ovine palatine tonsils, was used to fractionate rabbit and hog thymus. With respect to the chemical fractionation steps, yields of fractions, and optical and electrophoretic properties, extracts from hog and rabbit thymus were indistinguishable from similar extracts prepared from calf thymus. The study provides composition and yield data applicable to the thymus of a small mammal readily available in most laboratories.     

Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P

Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse-phase HPLC. Using an antiserum specific for the C-terminal hexapeptide amide of substance P, levels of 3.43 +/- 0.33 ng g-1 and 12.45 +/- 0.76 ng g-1 (mean +/- SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross-reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (less than 0.1%) were 12.46 +/- 0.47 ng g-1 and 7.20 +/- 0.37 ng g-1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse-phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co-eluting with the respective sulphoxides was higher in acidified ethanol extracts, and substance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse-phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

层析聚焦法分离纯化末端脱氧核苷酰转移酶及其抗血清制备和鉴定

 应用层析聚焦和Oligo(dT)-纤维素亲和层析相结合的方法,从小牛胸腺中分离纯化末端脱氧核苷酰转移酶(TdT)。纯化的TdT聚丙烯酰胺凝胶电泳呈一条区带,SDS-聚丙烯酰胺凝胶电泳分子量为24,000及26,000d的两条区带。此纯化TdT径戊二醛交联法,自身交联后免疫家兔,得到兔抗小牛TdT的单价抗血清,并进行了免疫学鉴定。     

Immunological evidence for the existence of H1-like histone in yeast

In view of the controversies about the existence of histone H1 in yeast we have reinvestigated the problem by studying yeast proteins extracted with perchloric acid and salt. Perchloric-acid-extracted proteins from whole cells contain only two fractions which comigrate with 'authentic' yeast high-mobility-group proteins (HMG) in both SDS and acid urea gels. These extracts show a considerable cross-reaction with anti-(calf thymus HMG) antiserum and do not react with antiserum to mouse liver H1. The isolation of 'authentic' yeast HMG by the standard salt/trichloroacetic acid procedure gives two types of preparations containing different numbers of protein bands. The poorer preparation reacts only with the anti-HMG antiserum whereas the richer preparation also gives considerable cross-reaction with the anti-H1 antiserum. Immunoblotting analysis performed on the salt-extracted proteins reveals the presence of three protein bands giving positive immunoreaction with the anti-H1 antiserum. The immunoreactive bands have electrophoretic mobilities close to that of the marker calf thymus H1 and similar to the mobilities of the presumptive yeast H1 fractions found by other authors.     

Application of capillary electrophoresis for DNA-protein binding tests

The combination of affinity chromatography and capillary electrophoresis (CE-SDS) has been found to be a useful tool to analyse populations of proteins which specifically bind to ssor dsDNA. Proteins were extracted from tissue, cytosol or nuclei of meristems of Pisum sativum seedlings and separated on cellulose column functionalized with ss-, dsDNA (calf thymus) and ssDNA (P. sativum) at 2M concentration of sodium chloride. Electropherograms of the crude protein extracts show two fractions of proteins specific for dsDNA (calf thymus) and three fractions specific for ssDNA (calf thymus). Four and five fractions of proteins specific for ssDNA (P. sativum) were identified in the material isolated from cytosolic and nuclear extracts, respectively. Both ds- and ssDNA (calf thymus) form complexes with ca. 4.0 % of the total amount of proteins, while ssDNA (P. sativum) binds to ca. 11.0 % of cytosolic and 5.0 % of nuclear proteins.     

Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA

All of the endogenous opioid peptides thus far identified are derived from three types of precursors, i.e. the corticotropin/beta-lipotropin precursor, preproenkephalin A and preproenkephalin B. Poly(A)-containing RNA from various bovine and porcine tissues has been subjected to blot hybridization analysis with the use of cDNA probes specific for the three opioid peptide precursors. Analysis with a corticotropin/beta-lipotropin precursor cDNA probe has revealed, in addition to the pituitary mRNA, a smaller hybridizable RNA species present in bovine extrapituitary tissues, such as the adrenal medulla, thyroid, thymus, duodenum and lung. The hypothalamus contains both these RNA species. DNA complementary to the smaller RNA species from the bovine adrenal medulla has been cloned. Analysis of the cloned cDNA, in conjunction with endonuclease S1 mapping of poly(A)-rich RNA from the adrenal medulla, has indicated that the smaller RNA species represents the 3'-terminal 712-729 nucleotides, excluding the poly(A) tail, of the pituitary corticotropin/beta-lipotropin precursor mRNA, having heterogeneous start sites. Analysis with a preproenkephalin A cDNA probe has shown the presence of hybridizable RNA in the bovine hypothalamus, duodenum and pituitary neurointermediate lobe in addition to the adrenal medulla. The hybridizable RNA species from all these tissues are indistinguishable in size. RNA hybridizable with a preproenkephalin B cDNA probe has been found in the porcine spinal cord and ileum besides the hypothalamus, and these RNA species exhibit an indistinguishable size. The results presented indicate that each opioid peptide precursor is synthesized in different tissues.     

Properties of inositol polyphosphate 1-phosphatase

We recently described inositol polyphosphate 1-phosphatase, an enzyme which cleaves the 1-phosphate from inositol 1,4-bisphosphate (Ins(1,4)P2) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) (Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 15946-15952). We have now purified the enzyme to homogeneity from calf brain. The enzyme hydrolyzes 50.3 mumol of Ins(1,4)P2/min/mg protein. The enzyme has an apparent mass of 44,000 daltons as determined both by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that it is monomeric. Lithium ions inhibit Ins(1,3,4)P3 hydrolysis uncompetitively with an apparent Ki of approximately 0.3 mM LiCl. Calcium inhibits hydrolysis of Ins(1,4)P2 and Ins(1,3,4)P3 equally, with approximately 40% inhibition occurring at 1 microM free Ca2+. Rabbit polyclonal antiserum against purified inositol polyphosphate 1-phosphatase was prepared which immunoprecipitates approximately 0.3 milliunits of activity/microliter serum (1 unit = 1 mumol of Ins(1,4)P2 hydrolyzed per min). This antiserum was used to determine the enzyme content in several bovine tissues, all of which had a similar intrinsic specific activity (i.e. approximately 0.3 milliunits/microliter antiserum). Tissues studied included brain, heart, kidney, liver, lung, parotid, spleen, testis, and thymus. Approximately 10-15% of the total inositol polyphosphate 1-phosphatase activity in calf brain homogenates remains in a particulate fraction; antiserum also binds 0.3 milliunits of membrane-associated activity/microliter antiserum. Thus, a single enzyme can account for Ins(1,4)P2 hydrolytic activity in the bovine tissues. Ins(1,3,4)P3 metabolism was also investigated in bovine tissue homogenates. Inositol polyphosphate 1-phosphatase accounts for greater than 80% of the hydrolytic activity in all tissues studied except brain, where inositol polyphosphate 4-phosphatase is the major enzyme that hydrolyzes Ins(1,3,4)P3. The apparent Km of inositol polyphosphate 1-phosphatase for Ins(1,3,4)P3 varies approximately 3-4-fold among the bovine tissues.     

Immunohistochemical localization of thymopoietin with an antiserum to synthetic Cys-thymopoietin28-39

Thymopoietin-containing cells in the thymus were identified immunohistochemically using murine antiserum generated by immunization with synthetic Cys-thymopoietin28-39 (Cys-TP28-39). human thymopoietin, This antiserum, previously shown to react with both bovine and human thymopoietin, gave reactivity restricted to cortical and medullary epithelial cells of bovine and human thymus. Monoclonal antibodies with reactivity restricted to native bovine thymopoietin did not react with tissue sections of bovine thymus; most likely the epitopes recognized by monoclonal antibodies are not expressed on the inactive precursor forms of thymopoietin within thymic epithelial cells.     

Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5.

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis-Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be -577 +/- 19 kJ.mol-1, 20.4 +/- 3.8 microm and 2.28 +/- 0.49 x 10-2 s-1, respectively, at 37.0 degrees C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A5 is of the same order of magnitude as that of lysozyme but several orders of magnitude lower than those of TaqI restriction endonuclease, NaeI endonuclease and BamHI endonuclease. By comparing the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. These results suggest that BLM-A5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5 follows Michaelis-Menten kinetics. Binding of BLM-A5 to calf thymus DNA is driven by a favorable entropy increase with a less favorable enthalpy decrease, in line with a partial intercalation mode involved in BLM-catalyzed breakage of DNA.     

Mammalian DNA ligases. Serological evidence for two separate enzymes.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.     

Distribution of vasoactive intestinal peptide, bombesin, and gastrin-cholecystokinin like peptides in the avian intestinal tract and brain

The distribution of vasoactive intestinal peptide (VIP), bombesin and gastrin-cholecystokinin in the chicken was studied by radioimmunoassay of tissue extracts. VIP was present in high concentrations in colon (186 +/- 29 pmol/g), cloaca (116 +/- 27 pmol/g), jejunum (97 +/- 14 pmol/g) and pancreas (15 +/- 3 pmol/g) but not detected in lung, liver or thymus. The highest concentration of bombesin was in the proventriculus (92 +/- 13 pmol/g), negligible in remaining gut but found in brain. Gel chromatography indicated two forms of bombesin: one form eluting with bombesin-14 and the other with gastrin releasing peptide. Gastrin-like immunoreactivity was found in low levels in the gut and brain. The concentrations were higher with an antiserum which cross reacted with the carboxy terminus common to gastrin-17 and CCK compared to a gastrin specific antisera (P less than 0.01). This suggests that the carboxy terminal region has been conserved during evolution. Each distribution pattern of bombesin, VIP and gastrin CCK is different, and distinct from that found in mammals, suggesting specific roles for these peptides in birds.     

Determination and comparative analysis of the catalytic subunit of adenosine 3',5'-cyclic phosphate-dependent protein kinase by an enzyme-linked immunosorbent assay.

A specific antiserum against bovine heart catalytic subunit was used for the determination of the catalytic subunit in an enzyme-linked immunosorbent assay. Under the conditions elaborated the assay has a lower detection limit for catalytic subunit of 0.25 pmol/ml. In crude bovine heart extracts the concentration of catalytic subunit was determined by this method to be 0.18 +/- 0.02 mumol/kg wet wt. The immunochemical comparison of various animal species and cells, including organisms like amoebae and yeast, shows the broad applicability of the assay and provides evidence that the catalytic subunit is a highly conserved molecule.     

Post-translational modification of bovine pro-opiomelanocortin. Tyrosine sulfation and pyroglutamate formation, a mass spectrometric study

The amino-terminal fragment of beta-lipotropin (i.e. beta-lipotropin (1-40)) and joining peptide portions of pro-opiomelanocortin have been purified from extracts of bovine posterior pituitaries. Peptides were purified using a combination of reversed-phase and ion-exchange batch extraction procedures followed by reversed-phase high performance liquid chromatography. beta-Lipotropin (1-40) was found to consist of four major components while joining peptide was found to consist of two major components. Fast atom bombardment-mass spectrometric analysis of the tryptic fragments of both peptides revealed that the observed heterogeneity could be explained in terms of post-translational modifications. beta-Lipotropin (1-40) was found to be sulfated at tyrosine residue 28 to an extent of about 50%. The tyrosine residue in beta-lipotropin (1-40) is situated within an amino acid sequence with a preponderance of glutamate residues. Sulfation of this amino acid residue is entirely compatible with the known primary structure requirements of the sulfotransferase enzyme located in the trans-Golgi fraction. Both beta-lipotropin (1-40) and joining peptide were found to have pyroglutamate at their amino termini to an extent of about 50%. The cDNA sequence for bovine pro-opiomelanocortin predicts the presence of glutamic acid at position 1 of both peptides. Pyroglutamate is normally formed through the cyclization of glutamine. This reaction is thought to be catalyzed by a pyroglutamate forming enzyme located within the secretory granule fraction. Under certain circumstances peptides with glutamate at their amino termini may act as substrates for this enzyme.     

双链核糖核酸免疫化学研究 Ⅰ.双链多聚[I]:多聚[C]的抗原性

用国产多聚肌苷酸和多聚胞嘧啶核苷酸(简称多聚[I]:多聚[C])制备成双链核糖核酸特异性抗血清,用环状沉淀和~3H标记的番茄花叶病毒双链核糖核酸测得抗血清效价分别为1:128和1:6400。用免疫琼脂双扩散、对流免疫电泳和放射免疫测定可测定出多聚[I]:多聚[C]的最低浓度分别为391ng、12.2/ng和10pg/ml抗血清和酵母、TMV、PVX 的单链核糖核酸、小牛胸腺DNA不反应。试验证明用国产多聚[I]:多聚[C]制备的抗血清,可有效地用干双链核糖核酸的免疫化学鉴定。由于在免疫双扩散和对流免疫电泳中抗血清能和微量双链核糖核酸反应并产生可见沉淀线,从而有可能利用这两种简便灵敏的方法测定病毒的双链RNA,单链RNA病毒的RF型RNA,以及双链RNA真菌病毒的筛选。     

Study of the thymus as a source of humoral factors stimulating antibody formation]

A fraction stimulating antibody formation in neonatal animals was obtained in separation of the calf thymus extract (1A) prepared by the method of Goldstein et al. on a column with Sephadex G-25 (fine). Testing of the activity of analogous fractions of the extracts of calf tonsils, spleen, lymph nodes and the liver led to a supposition that the thymus was the only source of specific humoral factor stimulating antibody formation, which could accumulate in the peripheral lymphoid formations. Low activity of tonsillar extracts failed to confirm the hypothesis on the role played by the tonsils as the central immunity organ similar to the bursa Fabricii.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.     

Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.     

Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition

Antiserum against calf thymus dUTPase was raised in rats by injection of the partially purified enzyme. The antiserum did not react with dUTPase purified from rat spleen, while antibody against rat spleen dUTPase partially reacted with calf thymus enzyme. Native molecular weight of the calf thymus dUTPase was estimated at 46,000 daltons by gel filtration, and the denatured form of the enzyme was about 22,000, as judged by immunoblot analyses using the antibodies. These results indicate that the calf thymus dUTPase is composed of two identical subunits.