Isolation of human pituitary prolactin

A process developed earlier for the extraction of human follitropin, lutropin, thyrotropin and growth hormone from homogenized frozen pituitaries provided a residue utilized for the isolation of prolactin. The isolation procedure involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B, molecular sieve chromatography on Sephadex G-100 Superfine, and ion-exchange chromatography on DEAE-Sepharose CL-6B using a convex gradient. The progressive purification was guided by radioimmunoassays. The final product was obtained in yields of 31 microgram/gland, and was equipotent with a pituitary preparation (VLS-3) supplied by the National Pituitary Agency (NIH, Bethesda, U.S.A.). Contamination hormones negligible (less than 0.05%). No heterogeneity of the isolated prolactin was observed by sedimentation-equilibrium analysis in the ultracentrifuge, by SDS electrophoresis in polyacrylamide gel or by molecular sieve chromatography in 6 M guanidine hydrochloride. These different techniques gave values in the range of 21 000-23 000 for the molecular weight of prolactin. In free zone electrophoresis, and also in polyacrylamide gel electrophoresis the prolactin preparation was, however, heterogeneous and resolved at alkaline pH into three distinct components. The former technique permitted isolation and assay of the components, indicating that they were all fully active.     

Isolation of Rat Pituitary Prolactin Isohormones Differing in Charge,Size, and Specific Immunological Activity

A method has been described for the isolation of three differently charged isohormones of rat prolactin from a discard fraction obtained after extraction of gonadotropins, thyrotropin and growth hormone from homogenized frozen pituitaries. The procedure involved extraction at pH 9.8, ammonium sulphate fractionation, molecular sieve chromatography on Sephadex G-100, and column electrophoresis in agarose suspension. The purification was monitored by radioimnunoassays and the recovered components were all found to possess a specific immunoactivity exceeding that of the standard preparation (RP-1) supplied by the NIAMDD, Bethesda, U.S.A. Increased acidity among these isohormones was found to be paralleled by significantly decreased immunopotency. Each component showed biological activity in radioreceptor assay.

A high degree of purity of the isolated components was shown by analytical electrophoresis in polyacrylamide gel. Sodium dodecyl sulphate electrophoresis in the same medium showed no size heterogeneity and yielded a value of approximately 25 000 for the molecular weight of the isohormones.

In addition a large form of prolactin, suggested to represent a dimer, was isolated by a further extraction step (pH 10.5) followed by molecular sieve chromatography on Sephadex G-100 and electrophoresis in agarose suspension. The large form was associated with both biopotency and immuno-potency. The electrophoresis resolved the prolactin activity into three or four immunoactive components. This pleomorphism of the large prolactin was confirmed by analytical polyacrylamide gel electrophoresis.

Amino acid analyses revealed a close similarity between the three monomers and the major dimeric form of the hormone.     

Purification and Characterization of High Molecular Weight Human Pituitary Prolactin

A large form of human prolactin (molecular weight 150 000–170 000) was purified from the residue remaining after extraction at neutral pH of homogenized frozen pituitaries. This purification involved extraction at pH 9.8, molecular sieve chromatography on Sepharose CL-6B, and hydrophobic interaction chromatography on pentyl-Sepharose 4B. The procedure was followed by radioimmunoassay. The large form of prolactin was prepared both from fresh and from long-term stored residues. In the latter case the final yield was considerably higher. By zone electrophoresis in agarose suspension the prolactin preparation was separated into four or five immunoactive components. In sedimentation equilibrium analysis in the ultracentrifuge, however, these isohormones showed heterogeneity, which was suggested to be caused by dissociation. Evaluation of data obtained from the bottom region of the cells gave molecular weight values of the components in the range of 160 000 – 180 000. One of the is hormones s further studied and exhibited bioactivity in the local crop-sac assay and showed an amino acid composition closely similar to that of the native monomer prolactin. The high molecular weight prolactin was partially dissociated by treatment with 50% ethylene glycol or 1 M propionic acid or 6 M guanidine hydrochloride. Molecular sieve chromatography in the presence of these dissociating agents, resolved the prolactin activity into three separate peaks. The most retarded fraction, which eluted in a position corresponding to that of native monomer prolactin was characterized by electrophoresis and amino acid analysis. The results were supporting evidence that the dissociation procedure gave a monomer which had a lower amide content than the native monomer. Furthermore, its specific immunoactivity was 2–3 times higher than the activity of the intact large form.     

Isolation of prolactin from lyophilized porcine pituitary glands]

The optimal conditions for lyophilization of porcine pituitary glands and isolation of pure prolactin from lyophilized preparation have been investigated. The isolation method consisted in the extraction of crude pituitary preparation with acidified acetone followed by precipitation of crude prolactin preparation (acid acetone powder) by increasing the concentration of acetone in the extract to 92%. Further purification of prolactin was achieved by fractional precipitation at varying pH values and gel filtration on Sephadex G-75 column in a pH 7.5 phosphate buffer. This final procedure resulted in obtaining the monomeric form of prolactin. The identity of the isolated hormone was confirmed by spectrophotometric and radioimmunological methods as well as by polyacrylamide gel electrophoresis.     

Isolation of Four Isomers of the 20,000 Dalton Variant of Human Pituitary Growth Hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20, 000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncova-lently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-1inked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Purification de l'alcool deshydrogénase de tomate par chromatographie d'affinité

Tomato alcohol dehydrogenase has been purified 99-fold by affinity chromatography on Blue Sepharose CL-6B with 37% yield. The enzyme so obtained is homogenous in polyacrylamide gel electrophoresis. By adding 20% glycerol to the extraction and purification buffers, an enzyme is obtained which is stable for several months at 4°. The molecular weight values determined by gel filtration (Sephadex G 200) and polyacrylamide gradient gel electrophoresis on one hand and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on the other, show that the enzyme exists in dimeric form.     

Isolation of four isomers of the 20,000 dalton variant of human pituitary growth hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20,000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncovalently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-linked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

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.     

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.     

大肠杆菌半乳糖结合凝集素的提取纯化

采用琼脂糖凝胶CL-6B（Sepharose CL-6B）亲和层析以及Sephadex G-75凝胶分子筛等对大肠杆菌（Esche-richia coli,E.coli）半乳糖凝集素进行了纯化。结果显示,目标蛋白经简单的步骤即可以得到纯化,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）及凝血实验证明纯化蛋白为E.coli半乳糖凝集素,蛋白提取回收率为11.4%。研究首次从E.coli蛋白提取液中分离得到纯的半乳糖凝集素,且此方法简单快捷,优越性明显。应用此方法将有利于微生物半乳糖凝集素的深入研究。     

Flavobacterium heparinum 3-O-sulphatase for N-substituted glucosamine 3-O-sulphate

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3-O-sulphate from 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose and 2-acetamido-2-deoxy-3-O-sulpho-D-glucose. The activity was purified 10 800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecylsulphate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56 000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulpho-D-glucose and 2-deoxy-2-sulphamido-3-O-sulpho-D-glucose as respective substrates. The purified 3-O-sulphatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N-acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2-fold enhancement of activity was effected by 0.002 mol dm-3 Ca2+. Inorganic phosphate and sulphate inhibited 3-O-sulphatase activity. The Km value of the N-acetylated substrate was determined to be 42 mumol dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulpho-D-glucose.     

Lack of interaction between hormonal proteins and synthetic carrier ampholytes (Ampholine)

Using ¹⁴C-labeled Ampholine, radioiodinated and unlabeled human growth hormone, and ovine prolactin, no significant degree of interaction could be detected by polyacrylamide gel electrophoresis and gel chromatography. Protein binding to excess Ampholine was negligible. Ampholine binding to excess protein was below the detection limit of 0.2 moles of Ampholine per mole of protein. Commercial Ampholine contains large molecular weight components that cannot be separated by gel chromatography from proteins in the 20–30,000-molecular weight range.     

Purification and characterization of anticomplement factor (cobra venom factor) from the Naja naja atra venom

Anticomplement factor (cobra venom factor) from the venom of Naja naja atra was purified by means of successive chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose CL-6B. The purified anticomplement factor was homogeneous as judged by polyacrylamide discontinuous gel electrophoresis at pH 9.4. The yield from 3.0 g of the crude venom was approx. 28 mg. The molecular weight was estimated to be about 156 000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was about 5.2. SDS-polyacrylamide gel electrophoresis of the anticomplement factor in the presence of dithiothreitol demonstrated that the molecule possesses three different polypeptide chains cross-linked covalently to one another by disulfide bridge(s). By SDS-polyacrylamide gel electrophoresis, the molecular weight of each subunit was determined to be approx. 77000, 47500 and 29 000, respectively. All subunits were stained with Coomassie brilliant blue G-250 and periodate-Schiff reagent, indicating these subunits to be glycoprotein. Distribution of the anticomplement factor in various snake venoms, which shows cross-reactivity against the anti-Naja naja atra anticomplement factor antiserum, was examined. From the results, all venoms belonging to cobra family in the Elapidae tested so far were found to contain such cross-reactivity.     

Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin

Described is a two-chromatographic-step preparative-scale technique for the purification of human prolactin from a frozen pituitary homogenate. The method utilizes hydrophobic interaction chromatography on the mildly hydrophobic adsorbent phenyl-Sepharose CL-4B and anion-exchange chromatography on DEAE-cellulose in the presence of acetonitrile. Human prolactin was solubilized at pH10.0 after a prior extraction of pituitaries at pH4.0, the acid pH being ineffective at solubilizing human prolactin but capable of solubilizing large amounts of interfering protein. An 11-fold increase in the potency of the solubilized human prolactin was achieved in this manner. Prolactin could be adsorbed to phenyl-Sepharose at low ionic strengths (I<0.01); few other proteins were adsorbed under these conditions. This is a demonstration of the hydrophobic nature of human prolactin. The amount of phenyl-Sepharose was limited to the minimum (35mg of protein/g of phenyl-Sepharose) necessary to adsorb human prolactin, further reducing the uptake of other pituitary protein. Desorption was achieved by using an acetonitrile gradient (0–30%, v/v), resulting in a purification of human prolactin of 85-fold and recovery of 78%. Acetonitrile (20%, v/v) was also included in all buffers for DEAE-cellulose chromatography, increasing the resolution and recovery of human prolactin, apparently by minimizing non-ionic interactions with the matrix. Prolactin (10mg) was recovered from 63g if pituitaries, an overall recovery of 58%. It was homogeneous by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, contained less than 0.1% somatotropin (growth hormone), on iodination demonstrated more than 95% binding to excess anti-(human prolactin) serum and could be displaced from anti-(human prolactin) serum in a manner indistinguishable from the serum of a patient with a human prolactin-secreting adenoma.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Purification to apparent homogeneity of inactive kallikrein from rat urine

Inactive kallikrein was purified from rat urine by a procedure including ammonium sulfate fractionation, DEAE cellulose chromatography, phenyl-Sepharose CL-4B chromatography, and gel filtration on Sephadex G-100 and Sephadex G-75 columns. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. This preparation migrated as a single protein band on a SDS-polyacrylamide gel and the molecular weight was 41000. The purified material underwent marked activation by trypsin, but not by deoxycholate, Triton X-100, SDS or acidification. These results indicate that the purified inactive kallikrein is the precursor rather than a complex with a substance binding to the active form of kallikrein.     

Purification and certain properties of pyruvate decarboxylase from bovine brain]

A procedure of isolation and purification of pyruvate decarboxylase (PDC) from bovine brain is worked out. 350-fold purified enzyme preparation was homogenous under polyacrylamide gel electrophoresis. Molecular weight of PDC from bovine brain was estimated to be 180 000 by means of gel chromatography through Sephadex G-200. The protein was eluted in two peaks (with molecular weight of 180 000 and 90 000 respectively). After the treatment of the enzyme preparation with 6 M guanidine chloride. Probably, partial dissociation of the enzyme molecule into two subunits takes place in this case. Data on paper chromatography confirmed that highly purified PDC preparations from bovine brain were isolated as apoenzyme, since they were almost free of TPP.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.