Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.     

Post-albumin, a foetal-specific rat plasma protein: Purification, physicochemical and immunological studies

1. The foetal-specific post-albumin of rat plasma was purified by electrophoresis on a Pevicon block. 2. The sedimentation coefficient, diffusion coefficient and molecular weight were determined for this protein and found to be similar to those of adult rat plasma albumin. 3. Foetal post-albumin and adult albumin were compared immunologically and shown, with rabbit antisera, to share no antigenic determinants, suggesting different genetic control of the production of each. 4. It is suggested that the disappearance of post-albumin in the newborn animal may result from the disappearance of haemopoietic tissue from the rat liver with advancing age.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.     

Thymidine kinase from Tetrahymena thermophila. Purification and immunological analysis

Thymidine kinase is an enzyme involved in DNA precursor metabolism and DNA replication. The synthesis of this enzyme is highly regulated during the cell cycle and the activity of the enzyme is also regulated by feedback inhibition. Genes encoding thymidine kinase have been extremely useful as selectable markers for introducing DNA into a number of cells. In order to study cell cycle regulation of thymidine kinase, the gene which encodes this enzyme, as well as aspects of DNA replication in the ciliated protozoan Tetrahymena thermophila, we have purified thymidine kinase from Tetrahymena. Two forms of thymidine kinase with native molecular masses of 59 kDa and 80 kDa have been identified and purified 6800- and 4600-fold, respectively. The 59-kDa enzyme, a homodimer of 30-kDa subunits, has been purified to near homogeneity and polyclonal antibodies have been raised against the 30-kDa subunit. Serological studies indicate that the two enzymes are antigenically distinct. The antibody against the Tetrahymena protein cross-reacts with a polypeptide in Chinese hamster ovary (CHO) cell extracts of 26 kDa which corresponds to the reported size of Chinese hamster thymidine kinase protein.     

Purification and immunological analysis of RNA polymerase II from Caenorhabditis elegans

We describe a rapid procedure for obtaining highly purified RNA polymerase II from the nematode Caenorhabditis elegans. The structure of the enzyme was examined by denaturing gel electrophoresis and found to consist of three large polypeptides (molecular weights 200,000, 175,000, and 135,000) and eight smaller polypeptides (molecular weights 29,500, 20,000, 16,000, 15,000, 13,000, 11,500, 10,500, and 9,500). As observed for the analogous enzyme from other organisms, the 175,000 polypeptide (II175) appeared to be a degraded form of the 200,000 polypeptide (II200). The structure of nematode RNA polymerase II closely resembles that of the corresponding enzyme from other animals. Four of its larger subunits shared antigenicity with Drosophila RNA polymerase II. Antibody raised against purified RNA polymerase II reacted with several enzyme subunits in "Western" blots of purified polymerase and impure enzyme fractions. Immunofluorescence staining was used to visualize RNA polymerase II in the nuclei of a nematode squash preparation and the nucleoplasm of cultured mammalian cells.     

Purification and properties of chicken prothrombin

Prothrombin was isolated from citrated chicken plasma. The isolation depends upon the elimination of an interfering substance closely adherent to chicken prothrombin by treatment with SrCO₃. Subsequent to this, the classical adsorption to barium citrate, chromatography on DEAE-cellulose, and gel filtration on Sephadex G-200 was carried out. Prothrombin purified by this method was found to have a specific activity of 1050 Iowa units (850 N.I.H. thrombin units) per mg. Recovery from plasma averaged 40%. Molecular weight by Sephadex G-200 chromatography was 73,000 ± 5,000 and by dodecyl sulfate sodium salt acrylamide gel electrophoresis 70,000 ± 5,000. A stable dimer of M_r 138,000 was observed in some preparations. The isoelectric pH in both acetate and phosphate buffers (μ = 0.1) was 3.95. Rabbit antibody to chicken prothrombin evidenced a single line by immunoelectrophoresis against purified antigen and chicken plasma.     

Purification and isoelectric heterogeneity of chicken tyrosinase

Tyrosinase (EC 1.14.18.1) was purified from regenerating chicken feathers. Most of the enzyme activity was in the insoluble fraction, which was solubilized with 0.5% sodium cholate. Solubilized tyrosinase showed multiple forms on isoelectric focusing. The isoelectric points had the following pI values: 5.06, 4.83, 4.68, 4.56, 4.44, 4.32, 4.24, 4.14, 4.06 and 3.97. This tyrosinase fraction was subjected to trypsin (EC 3.4.21.4) cleavage, Sephacryl S-200, hydroxylapatite and DEAE-cellulose chromatography. Purified enzymatically active tyrosinase also showed multiple forms. Their isoelectric points were: 4.23, 4.14, 4.06, 3.99 and 3.91. Each active form had almost the same molecular weight, estimated at 66 000. Staining for 1,2-diol groups of glycoproteins and neuraminidase (EC 3.2.1.18) treatment suggested that chicken tyrosinase is a glycoprotein. The enzyme showed both dopa(L-3,4-dihydroxylphenylalanine) oxidase activity and tyrosine hydroxylase activity.     

Purification and isoelectric heterogeneity of chicken tyrosinase

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5' end of the molecule. This group of tRNAs includes E. coli tRNANfmet, tRNAAla1, tRNALeu1, or Leu2, and tRNASer3 (Group 1). In each case N2-methylguanine and N2,N2-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N2-guanine methyltransferase II (S-adenosyl-L-methionine : tRNA (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50 degrees C for min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preparations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N2-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Goat testis calmodulin: Purification and physicochemical characterization

Calmodulin has been purified in large quantities from goat (Capra hiscus) testis. The procedure includes heat treatment, hydrophobic interaction chromatography, and gel filtration. Goat testis calmodulin closely resembles other mammalian testis calmodulin studied so far. The protein has an extinction coefficient value (E _1cm ^1% ) of 2.09 at 280 nm, a Stokes radius of 23.2 Å at 0.15 M KCl, and a frictional ratio of 1.38. Ca²⁺, and Tb³⁺ binding studies demonstrate that the protein has four Ca²⁺-binding sites with aK _d of 52.5 µM. Goat testis calmodulin shows close similarity to other calmodulins in the amino acid composition and in demonstrating an altered migration on SDS/PAGE upon Ca²⁺ binding. The protein also exhibits anomalously high values for molecular weight and Stokes radius as determined from the analytical gel chromatography and a change in its elution volume with the change of salt concentration in the eluant. These results have been discussed in view of the recently available knowledge from the crystallographic studies of rat testis calmodulin.     

Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological protperties.

Cells of Pseudomonas putida NP, Pseudomonas species (NCIB 9816), and a Nocardia species, after growth on naphthalene as sole source of carbon and energy, contain a nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that oxidizes cis-dihydrodiols of mono- and polycyclic aromatic compounds. Similarly, cells of a strain of P. putida biotype A, when grown either on toluene or benzene vapors, were found to contain a dehydrogenase that oxidized dihydrodiols of aromatic hydrocarbons with cis stereochemistry and required NAD+ as an electron acceptor. In all these cases, no enzymatic activity was detected when trans-naphthalene dihydrodiol was used as a substrate. Purified cis-naphthalene dihydrodiol dehydrogenase was injected into rabbits to obtain antibodies. Physiocochemical and immunological properties of cis-dihydrodiol:NAD+ oxidoreductases from four different organisms were examined. Kinetic analysis showed that, in all the cases, enzymes exhibited higher affinity for cis-dihydrodiols than for NAD+ and had pH optima between 8.8 and 9.0. except in the case of the enzyme from Nocarida sp., which showed maximum activity at pH 8.4. Molecular-weight determination of the dehydrogenases from the four different organisms by gel filtration on a Sephadex G-200 column gave values ranging from 92,000 for the enzyme from Nocardia sp. to 160,000 for that from P. putida biotype A. All the dehydrogenases, except the one from Nocardia sp., exhibited immunological cross-reaction with the antibodies prepared against the enzyme purified from P. putida NP.