High molecular weight complexes of folic acid in mammalian tissues

Twenty-four hours after injection of tritiated folic acid into normal rats, a large amount of labeled folates were found to be associated with the high molecular weight fraction of liver, kidney and intestine. The bound folate was associated with three fractions in the liver cell supernatant having approximate molecular weights of ? 350,000, 150,000 and 25,000 daltons, respectively. A fourth fraction which had an approximate molecular weight of 90,000 daltons was isolated from the liver nuclear fractions. The bound folates associated with these fractions were almost exclusively polyglutamate forms.     

Repetitive DNA of Higher Plants: SEPARATION OF FRACTIONS OF DIFFERENT COMPLEXITY FROM JERUSALEM ARTICHOKE TISSUES BY REASSOCIATION KINETICS

Jerusalem artichoke DNA, extracted from resting rhizomes, wasanalysed by the reassociation kinetics of sheared denaturedfragments. Various fractions were isolated according to theirreassociation rates. The slowest fractions consist of unrepeatedor single-copy DNA which makes up 55 per cent of the total DNA.The total sequence length of the haploid genome is estimatedas corresponding to 0.23?10¹² daltons. Among the fractions ofrepeated DNA isolated, two were relatively homogeneous withan average complexity of 2?10⁸ and 10⁹ daltons respectively.     

Characterization of distinct tyrosine-specific protein kinases in B and T lymphocytes

Lymphocyte membrane fractions from both normal and neoplastic sources exhibit tyrosine-specific protein kinase activity. The molecular weights of the endogenous substrates phosphorylated on tyrosine residues differ in B and T cells. To further characterize membrane tyrosine phosphorylation in the two major classes of lymphocytes, the tryptic phosphopeptides of their endogenous substrates were compared and the sensitivity of the kinases to inhibition by N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) was determined. The two major B cell substrates (61,000 and 55,000 daltons, p61 and p55) were gel purified after phosphorylation and exhaustively digested with trypsin. Separation by reverse phase high pressure liquid chromatography demonstrated that these two substrates had two identical phosphotyrosine containing tryptic phosphopeptides. p61 had an additional phosphotyrosine site. Parallel analysis of the two T cell substrates (64,000 and 58,000 daltons, p64 and p58) showed that they also contained two phosphotyrosine sites that were identical. However, the tryptic phosphopeptides from the B and T cell substrate pairs were clearly distinct suggesting that they arise from different gene products. When B and T cell membrane fractions were preincubated with TLCK (21 degrees C, 30 min) a dose-dependent decrease in p64 and p58 phosphorylation resulted. p61 and p55 phosphorylation was not affected at concentrations up to 10 mM TLCK. Tyrosine-specific kinase activity was also assessed by measuring phosphorylation of a tyrosine containing synthetic peptide. The kinase activity of T cell plasma membrane fractions was inhibited by TLCK; the B cell activity was unaffected. The results suggest that membrane fractions from normal and some neoplastic B and T cells have at least two different tyrosine-specific kinases.     

Chemical characterization of two extracts used in the determination of available soil nitrogen

Summary Two soil extracts used for chemical indexes for N availability, 0.01M NaHCO₃ and boiling 0.01M CaCl₂, were analyzed in effort to learn more about the nature of the extracted organic matter (O.M.). The two extracts appeared to remove different fractions of the soil O.M. A study of five soils showed that the C/N value of the NaHCO₃ extract (following decarbonation) was significantly higher than that of the total soil O.M.; while the C/N value in the boiling CaCl₂ extract was not significantly different from that in the soil O.M. There was also significant variation in C/N values among soils for the boiling CaCl₂ extract. The extracts of three soils were analyzed for apparent molecular weight distribution using gel filtration and the results compared to those for base-extracted humic substances. Almost all the molecules in the extracts had apparent molecular weights less than 21,000 daltons while 21 to 47% of the humic substances from the same soils (extracted with 0.5M NaOH) had molecular weights greater than 21,000 daltons. In the boiling CaCl₂ extract, 78 to 87% of the humic substances had apparent molecular weights less than 1,000 daltons, whereas with the NaHCO₃ extract, 42 to 83% of the humic substances were in the 1,000 to 21,000 dalton range. Forty-three to 92% of the N extracted by the NaHCO₃ was in protein form, and 8 to 30% was ninhydrin-detectable. In the boiling CaCl₂ extract 25 to 30% of the extracted N was ninhydrin-detectable. For the same 10 soils, ninhydrin-detectable N values of the boiling CaCl₂ extract appeared closely related to greenhouse and field relative N uptake, while the ninhydrin-detectable N values of the NaHCO₃ extract appeared unrelated to both. The protein N and protein in plus ninhydrin-detectable N values of the NaHCO₃ extract were closely related to greenhouse relative N uptake only. The results of this study indicated that specific fractions of the soil O.M. were being extracted by the two solutions and that significant differences existed in the chemical nature of the two extracts. Paper No. 6175 of the J. Ser. of the Pennsylvania Agric. Exp. Stn. Authorized for publication Jan. 26, 1981.     

Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

抗尿激酶单克隆抗体识别相应抗原决定簇的研究

 尿激酶是一种纤溶酶原激活剂,临床上用于治疗血栓。为了有效地用单克隆抗体亲和柱纯化尿激酶,我们对一组抗尿激酶单克隆抗体识别相应抗原决定簇的特性进行了研究。Western Blotting试验表明:S_(13)、S_(26)、N_(14)、N_(30)、N_(17-2)、N_(34)、N_(36)七个单克隆抗体主要抗54000道尔顿的高分子量尿激酶(HUK)。除N_(30)外,其余抗体还同时不同程度地抗33000道尔顿的低分子量尿激酶(LUK)。N_(30)除识别HUK外,还识别分子量为18000道尔顿的多肽链。竞争性结合试验证明:七个单克隆抗体分别抗五个不同的抗原决定簇,但它们都不抗尿激酶的活力中心。     

MONOAMINE OXIDASE (EC 1.4.3.4): ISOLATION AND CHARACTERIZATION OF MULTIPLE FORMS OF THE BRAIN ENZYME

Monoamine oxidase (MAO) in crude mitochondrial preparations from rat brain was solubilized, and different MAO-active fractions were separated by agarose columns and by Sephadex electrophoresis. Any combination of these techniques yielded at least three fractions possessing MAO activity as measured by assays using radioactive serotonin and benzylamine as substrates. The molecular weight of one of the MAO forms was found to be approximately 400,000 daltons while another was at least 1.5 × 10⁶ daltons. The crude mitochondria1 MAO was inhibited by [¹⁴C]-labelled pargyline and then solubilized and the radioactivity of the soluble and particulate MAO was compared to the enzyme activity found in the soluble and particulate fractions. Our studies suggest that appreciable MAO activity is lost upon solubilization and that the conformation of MAO may be altered.     

Immunochemical analysis and protective properties of components of Pseudomonas aeruginosa mucus with different molecular weights

P. aeruginosa slime has been separated into fractions XM-300 (3 X 10(5) daltons and more), XM-100 (1 X 10(5) = 3 X 10(5) daltons), PM-30 (3 X 10(4) = 1 X 10(5) daltons) and PM-10, (1 X 10(4) = 3 X 10(4) daltons) by ultrafiltration. The high-molecular slime components (3 X 10(4) daltons and more) have been found to be serologically more active than the low-molecular components (1 X 10(4) = 3 X 10(4) daltons). As shown in experiments on mice, both high-molecular toxic and low-molecular nontoxic slime components have protective activity, but the high-molecular components are more active than the low-molecular ones. The slime components stimulate the formation of immunity to homologous and partially heterologous P. aeruginosa strains in mice. Antigenic relationship between the slime components (especially the high-molecular ones) and the corresponding lipopolysaccharides has been noted.     

Molecular characterization of an insect genome: Chironomus thummi.

DNA extracted from Chironomus thummi larvae was studied by isopycnic centrifugation in CsCl, thermal denaturation and DNA-DNA reassociation techniques. The mean G+C content of the C. thummi DNA is 28-29% as indicated both by centrifugation in CsCl and thermal denaturation. According to optical reassociation analysis of total DNA and of isolated DNA fractions the C. thummi genome is composed of at least four components. About 80% of the DNA is classified as unique with a kinetic complexity of nearly 7 X 10(10) daltons. 6-8% intermediate DNA exhibits a kinetic complexity slightly above 10(8) daltons with a mean repetition frequency of 35. 11-13% fast-reassociating DNA has a kinetic complexity slightly above 10(6) daltons with a mean repetition frequency of 6000. 3-5% of the DNA cannot be properly studied by the optical reassociation technique and probably contains inverted repeats. The thermal denaturation behaviour of isolated DNA fractions indicated that most of the repetitive sequences in the C. thummi genome are tightly interspersed.     

Correlation and modulation of Ehrlich ascites tumor growth with tumor-plasma IgA

Plasma IgA level of Ehrlich ascites tumor bearing mice showed correlation with progress of tumor growth. In PAGE analysis total plasma IgA separated into 3 major bands corresponding to mol. wt. > or = 669,000 daltons, identical to 443,000 daltons and between 443,000 and 150,000 daltons. All the three bands increased gradually with progress of tumor growth upto day 14 and then declined on day 16. Total plasma IgA isolated by anti-IgA affinity chromatography when adoptively transferred to mice inhibited tumor growth. Affinity-purified plasma IgA separated into three major peak fractions after Sephadex G-200 column chromatography which corresponded with the bands of IgA on PAGE analysis. Three Sephadex G-200 IgA fractions when adoptively transferred to tumor-bearing mice showed effect different from total IgA. High mol. wt. IgA fraction (> or = 669,000 daltons) inhibited tumor growth whereas medium mol. wt. fraction (identical to 443,000 daltons) enhanced tumor growth. The low mol. wt. IgA fraction (< 443,000 and > 150,000 daltons) had no significant effect on tumor growth. The high mol. wt. IgA fraction enhanced tumor killing ability of peripheral blood lymphocytes (PBL) and peritoneal macrophages of tumor bearer in vitro. Medium mol. wt. IgA fraction inhibited tumor-killing ability of PBL in vitro but had no significant effect on peritoneal macrophages. The low mol. wt. IgA fraction showed a mild enhancing effect on tumor-killing ability of PBL but had no significant effect on peritoneal macrophages. The results established importance of IgA in tumor growth regulation and its therapeutic potentiality. The results indicated that tumor growth modulation by tumor plasma IgA is also mediated by its effect on cellular anti-tumor immune factors of the host.     

Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes

(³H)Cytochalasin B has been photoincorporated into membrane fractions of the human erythrocyte, Rous sarcoma virus-transformed chicken embryo fibroblast and rat adipocyte. Identification of D-glucose sensitive cytochalasin B binding sites was achieved by photolyzing membranes with radioligand in the presence of 0.5–0.7M D- or L-glucose. In the erythrocyte the major labeled bands on SDS-polyacrylamide gels were at 55,000 and 46,000 daltons. In the virus-transformed fibroblasts a major labeled band was at 55,000 daltons, and in adipocyte microsomal membranes, peaks at 50,000 and 45,000 daltons were observed. Binding characteristics of these polypeptides suggest that they are the putative glucose transport proteins in these three cell types.     

Protective antigens of the vaccinia virus

The comparison of the polypeptide composition of 3 vaccinia virus strains, L-IVP, B-51 and CM-63, has revealed that strains L-IVP and B-51 are similar in their polypeptide composition, while in strain CM-63 capsid polypeptide with a molecular weight of 34000 daltons is absent or has altered electrophoretic mobility. As the result of the isolation of vaccinia envelopes (from strain L-IVP) and the electrophoretic separation of their polypeptides in plates with polyacrylamide gel 10 polypeptides have been obtained in 7 fractions, each containing 1 or 2 polypeptides. The immunization of rabbits with individual fractions has demonstrated that the formation of virus-neutralizing antibodies is induced mainly by 4-5 polypeptides in 3 fractions, having the highest molecular weight (54000-31000 daltons) and constituting about 19% of all proteins in the whole virion. The low-molecular envelopes polypeptides have been found to play no essential role in inducing the formation of virus-neutralizing antibodies. The highest antibody titers (1: 15625) have been detected in antisera to the preparations of whole vaccinia virus envelopes.     

Purification and properties of tyrosyl tRNA synthetase of rat liver.

Purification of rat liver tyrosine tRNA synthetase yields two protein fractions A and B and both fractions are required for charging of tyrosine to tRNAtyr. Fraction B catalyzes the activation of tyrosine. Fractions A and B have been purified to near homogeneity and they are composed of single polypeptide chains of 62,000 daltons each. Gel filtration studies suggest a molecular weight of 120,000 for the synthetase.     

Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins

A technique is presented which enables one to measure the extent to which a protein enters and accumulates in the nucleus of the frog oocyte. In this method, the protein, labeled with 125-I, is microinjected into the oocyte. After incubation, the oocyte is manually enucleated and the radioactivity in the nucleus and cytoplasm is determined. Using this technique, proteins lighter than 20,000 daltons were found to enter the nucleus and completely equilibrate between the nucleus and cytoplasm within 24 h. The entry of proteins heavier than 69,000 daltons was severely hindered. Histones and histone fractions entered as quickly as other small proteins, but, in contrast to these proteins, they accumulated in the nucleus to different extents, depending on the total amount of histone injected into the oocyte and the identity of the histone. Evidence is presented that histone fractions compete with each other for accumulation in the nucleus.     

Two homogeneous myosins in body-wall muscle of Caenorhabditis elegans

Myosin purified from the body-wall muscle-defective mutant E675 of the nematode. Caenorhabditis elegans, has heavy chain polypeptides which can be distinguished on the basis of molecular weight. On SDS-polyacrylamide gels, bands are found at 210,000 and 203,000 daltons. This is in contrast to myosin from the wild-type, N2, which has a single heavy chain band at 210,000 daltons. Both heavy chains of E675 are found in body-wall muscle (Epstein, Waterston and Brenner, 1974).When native myosin from E675 is fractionated on hydroxyapatite, it is separated into myosin containing predominantly one or the other molecular weight heavy chain and myosin containing a mixture of the heavy chains. Comparison of the CNBr fragments of myosin that contains predominantly 210,000 dalton heavy chains with those of myosin that contains predominantly 203,000 dalton heavy chains reveals multiple differences. These differences are not explained by the difference in molecular weight of the heavy chains, but may be explained if each type of heavy chain is the product of a different structural gene. Furthermore, because there are fractions which exhibit >80% 210,000 or >80% 203,000 dalton heavy chain, there is myosin which is homogeneous for each of the heavy chains.Although N2 myosin has only a single molecular weight heavy chain, it too is fractionated by hydroxyapatite. By comparing the CNBr fragments of different myosin fractions, we show that N2, like E675, has two kinds of heavy chains.E190, a body-wall muscle-defective mutant in the same complementation group as E675, is lacking the myosin heavy chain affected by the e675 mutation. This property has allowed us to determine by co-purification of labeled E190 myosin in the presence of excess, unlabeled E675 myosin that most, if not all, of the myosin that contains two different molecular weight heavy chains is due to the formation of complexes between homogeneous myosins and not to a heterogeneous myosin.     

Localization of proteins controlling motility and chemotaxis in Escherichia coli.

Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.     

Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

Two-factor requirement for murine megakaryocyte colony formation

WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.     

Heavy and light forms of some aminoacyl-tRNA synthetases in fraction X,microsomes and cytosol of rabbit liver

Summary Aminoacyl-tRNA synthetase activity for alanine, glutamic acid, lysine and phenylalanine was studied in the three subcellular fractions of rabbit liver: fraction X, microsomes and cytosol. From 60 to 80% of the enzyme activities were found in fraction X and microsomes. Fraction X was especially rich in the synthetase activities. By means of gel chromatography, heavy (over 10⁶ daltons) and light (below 480 × 10³ daltons) forms of lysyl- and phenylalanyl- but only light ones of alanyl- and glutamyl-tRNA synthetase activities were found in all the subcellular fractions studied. It is concluded that in higher organisms (mammals) all aminoacyl-tRNA synthetases, at least in part, are associated with cell structural constituents.Abbreviations ALA, GLU, LYS, PHE alanyl-, glutamyl-, lysyl-, phenylalanyl-tRNA synthetase - PMSF phenylmethylsulfonyl fluoride - BSA bovine serum albumin