Partial separation of the pituitary proteins of Labeo umbratus, Smith (mudfish)

L. umbratus pituitary glands were partially separated by means of preparative polyacry-lamide electrophoresis and chromatography on DEAE-cellulose and Sephadex G-50. Eight fractions were obtaìned and it was found that fraction 1 contained potent adrenocorticotropic stimulating activity, with a molecular weight in the region of 6000. Fractions 3 and 4 contained potent gonadotropic and a lesser degree of exopthalmic producing activity, with molecular weights in the region of 14 000 to 20 000. Fraction 6 displayed pigeon-crop stimulating activity, with a molecular weight above 30 000. Fractions 7 and 8 displayed M.S.H. and vasopressinoxytocic activity respectively, with a molecular weight in the region of 5500–6500. Fractions 2 and 5 could not be identified. The fractions seemed to be glycoproteins and the results are discussed in relation to previous findings.     

Heterogeneity of circulating prolactin in the bitch

Different molecular forms of circulating prolactin (PRL) are known to occur in several species. As no such information was available in dogs, we assessed the molecular profile of circulating PRL in bitches. Pooled sera from covertly (CTRL) and overtly pseudopregnant (PSPT) diestrous bitches with high or low (> 10 or < 10 ng x mL(-1), respectively) serum PRL (measured by ELISA) were analyzed by Sephadex G-100 and Concanavalin A-Sepharose column chromatography. Four serum PRL fractions were identified and termed big-big, big (> 67 kDa), native (23 kDa) and fragmented (< 20) kDa) PRL. The percentages of these fractions were roughly similar in CTRL and PSPT animals, irrespective of their serum PRL levels (higher in PSPT than in CTRL bitches). A large proportion of glycosylated PRL (between 69 and 100%) was also detected in these sera. We conclude that in dogs, circulating PRL occurs in multiple molecular forms, whose relative abundance is comparable in covertly and overtly pseudopregnant bitches.     

Human leukocyte inhibitory factor (LIF): two distinct molecular species.

Human leukocyte inhibitory factor or LIF was generated in vitro by stimulating blood lymphocytes with concanavalin A (Con A). The control and Con A active supernatants were partially purified by gel filtration on Sephadex G-100. The fraction containing LIF (68,000 daltons) activity was then subjected to isoelectric focusing (pH 3 to 10 ampholines) in a sucrose gradient. Two LIF activities were reproducibly recovered by this procedure. One molecular form was found to have an isoelectric point of approximately pH 5.0 and the other approximately pH 8.5. Both molecular species were rechromatographed on Sephadex G-75 and found to have the same apparent m.w. (68 to 75,000). Furthermore, the biologic activity of both factors was destroyed after treatment with diisopropylphosphofluoridate, suggesting that they may be esterases.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Alanine aminotransferase and glycine aminotransferase from maize (Zea mays L.) leaves.

Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio.     

Identification of polysaccharides from pericarp tissues of litchi (Litchi chinensis Sonn.) fruit in relation to their antioxidant activities

A large number of polysaccharides are present in the pericarp tissues of harvested litchi fruits. A DEAE Sepharose fast-flow anion-exchange column and a Sephadex G-50 gel-permeation column were used to isolate and purify the major polysaccharides from litchi fruit pericarp tissues. Antioxidant activities of these major polysaccharide components were also evaluated. An aqueous extract of the polysaccharides from litchi fruit pericarp tissues was chromatographed on a DEAE anion-exchange column to yield two fractions. The largest amount of the polysaccharide fraction was subjected to further purification by gel filtration on Sephadex G-50. The purified product was a neutral polysaccharide, with a molecular weight of 14 kDa, comprised mainly of 65.6% mannose, 33.0% galactose and 1.4% arabinose. Analysis by Smith degradation indicated that there were 8.7% of (1-->2)-glycosidic linkages, 83.3% of (1-->3)-glycosidic linkages and 8.0% of (1-->6)-glycosidic linkages in the polysaccharide. Furthermore, different polysaccharide fractions extracted and purified from litchi fruit pericarp tissues exhibited strong antioxidant activities. Among these fractions, the purified polysaccharide had the highest antioxidant activity and should be explored as a novel potential antioxidant.     

Purification and characterization of exudate gelatinases in the chronic-phase of carrageenin-induced inflammation in rats

Gelatinases have been purified from the exudate in the chronic-phase (day 7) of carrageenin-induced inflammation in rats. The day-7 exudate gelatinases gave two peaks on Sephadex G-150 gel filtration, the initial step of the purification. The molecular weights of the gelatinases corresponding to the two peaks were about 300 kDa (HMW fraction) and about 110 kDa (LMW fraction), respectively. The gelatinase in the HMW fraction has been purified to homogeneity; the purified gelatinase gave a single band corresponding to a molecular weight of 57 kDa on both SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-gelatin PAGE. On the other hand, the gelatinase purified from the LMW fraction was found to consist of three species, with molecular weights of 66, 64, and 57 kDa, as judged on SDS-gelatin PAGE. Granulation tissue-derived fibroblasts in culture mainly produced the 64-kDa species, which was converted to a 57-kDa species on treatment with 4-amino-phenylmercuric acetate, while rat macrophages and polymorphonuclear leukocytes mainly secreted the 96-kDa species. These results suggest that exudate gelatinases are largely produced by fibroblasts in granulation tissue and that they bind to exudate proteins, resulting in the formation of complexes with molecular weights of about 300 kDa and about 110 kDa. The gelatinases purified from the HMW and LMW fractions are metalloproteinases, as judged from the results of inhibitor experiments. Both the gelatinases degraded gelatin, but showed to proteolytic activity toward alpha-casein or type I collagen. Type IV collagen was degraded at 35 degrees C by the gelatinases purified from the LMW fraction but not by that from the HMW fraction.     

Purification and characterization of E. histolytica antigen for diagnosis of amoebiasis

Entamoeba histolytica soluble crude antigen was fractionated by gel filtration on Sephacryl S-300 into four fractions, viz. F1(669 kDa); F2(51.2 kDa); F3(25.1 kDa) and F4(10.5 kDa). F1 fraction was observed to be more sensitive and specific for the detection of antibody in amoebiasis than the crude and other fractions of purified antigens employing IHAT and ELISA. ELISA was found to be better than IHAT since it could detect antibody in the sera (3/6) of asymptomatic cyst passers. The cross reaction of crude antigen with toxocariasis (1/4) and toxoplasmosis (2/5) sera were associated with F4 fraction. F3 and F4 were having low molecular weight and were not sensitive in detection of antibody in amoebiasis. Biochemical characterization revealed glycoprotein nature of the specific (F1) antigen fraction.     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Solubilization of membrane-bound acid alpha-glucosidase by proteolysis

The membrane-bound acid alpha-glucosidase was purified partially (400-fold) from human placenta by solubilization with trypsin, concanavalin A-Sepharose chromatography, Ultrogel AcA-34 gel filtration, and Sephadex G-100 affinity chromatography. Two molecular forms of the enzyme were found in the final preparation of the purified enzyme. They were identical in molecular weight with a precursor (110 kDa) and an early intermediate form (105 kDa) of this enzyme. Also direct incubation of the membrane fraction without trypsin resulted in a release mainly of the 105 kDa form, which was inhibited by N-ethylmaleimide, but not by leupeptin, pepstatin or phenylmethylsulfonylfluoride. It was concluded that the precursor of acid alpha-glucosidase is an intrinsic membrane protein, which is transported into lysosomes after solubilization by proteolysis.     

Purification and Characterization of B2 Bradykinin Receptor from Rat Uterus

Abstract: A B₂ bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G-50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500-fold, was then cross-linked to ¹²⁵l-Tyr⁰-BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two-dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80–120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B₂ receptor. The partially purified and the crude solubilised B₂ BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo-Tyr⁰-BK, D-Phe⁷-BK, and des-Arg⁹-BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B₂ BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5–4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.     

Extraction and partial purification of prolactin-release stimulating factor in bovine hypothalami.

Partial purification of prolactin-release stimulating factor (PRF) was performed by Sephadex G-25 gel filtration of bovine hypothalamic extracts. PRF activity was evaluated on the basis of the measurement of immunoreactive prolactin released from the isolated rat hemipituitary in vitro. PRF activity was found in the fractions with Kav=0-0.49 and prolactin-release inhibiting activity was also detected in the fractions with Kav=0.69-0.89. The dose-response relationship was established between the partially purified PRF and its activity. The elution position of the partially purified PRF preceded that of TRH on Sephadex G-25. TRH at the dose of 100 nM stimulated the release of TSH in vitro, but not the release of prolactin. These results may indicate that there exists PRF with a relatively high molecular weight in the bovine hypothalamus.     

Growth inhibitors in plasma derived human serum

Summary It was reported previously that plasma derived human serum (PDS) inhibited the growth of cells established from malignant human breast tissues and the MCF-7 cell line but did not inhibit the growth of cells from nonmalignant mammary tissues, including the HBL-100 cell line. Plasma derived human serum was fractionated in the current study by molecular sieve chromatography on Sephadex G-100 in an effort to characterize the factor(s) responsible for inhibition. Plasma derived human serum contained several growth inhibitory fractions, which were designated G-1, G-2, G-3, and G-4. The G-1 was associated with the lipoproteins and immunoglobulins of serum. The lipid portion of G-1 inhibited the growth of both MCF-7 and HBL-100 cells, whereas the protein fraction contained a low activity factor directed against MCF-7 cells only. The G-2 also inhibited MCF-7 cell growth at a low specific activity and was separated in the serum albumin fraction. The MCF-7 inhibitory activity in the G-3 fractions from individual donors fluctuated with the level of activity in the starting sera. The cell specific G-3 components were purified further by Sephadex G-100 superfine chromatography and gel electrophoresis. A tentative molecular weight of 50,000 was assigned to the G-3 inhibitor. The G-4 fraction consisted of small molecular weight materials migrating in advance of the column volume, which inhibited the growth of both cell lines. This investigation was supported by Grant PDT-140 from the American Cancer Society, Inc., and PHS Grant CA30284 awarded by the National Cancer Institute, Bethesda, Maryland.     

Isoenzymes of Starch Phosphorylase in Triticale

Starch phosphorylase (α-1,4 glucan: orthophosphate α-D-glucosyl transferase, EC 2.4.1.1) was partially purified from triticale by ammonium sulphate fractionation and chromatography on Sephadex G-200. Three active fractions were obtained, which differed in their kinetic properties and activities in the presence of certain glycolytic and Krebs cycle intermediates. These fractions may have specific functions in starch synthesis during grain development. The isoenzymic fraction Ell, in particular, may have a regulatory function by controlling formation of primers from oligosaccharides.     

The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1

Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions.     

Purification and characterisation of phenoloxidase from amphioxus Branchiostoma belcheri tsingtauense

Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Characterization and partial purification of a neurotrophic factor for cerebrocortical neurons from chicken cerebral extract

The survival of cerebrocortical neurons from 6 to 8-day-old chick embryos was investigated in a serum-free hormone-supplemented medium. The addition of cerebral extract promoted the survival of cortical neurons in a dose-dependent manner, but induced almost no neurite outgrowth. The trophic activity was higher in the adult cerebrum than in the embryonic cerebrum. The tropic factor was partially purified from adult chicken cerebrum, and the molecular weight of the factor was estimated to be about 60 kDa. The activity for survival factor was fairly resistant to heat or trypsin treatment. When the partially purified sample was, however, treated with trypsin (1 mg/ml) for 20 h and applied to a TSK G2000 SW gel filtration column, the activity moved from 60 to 70 kDa untreated active fractions to fractions with about 10 kDa. These physicochemical properties of the survival factor suggest a new class of macromolecular trophic factors in the brain.     

Partial purification and characterization of Gigantocotyle explanatum somatic antigens

Soluble extracts of Gigantocotyle explanatum, isolated from the liver of buffalo Bubalus bubalis were fractionated on Sephadex G-200 columns. Nine major fractions referred to as F1, F2, F3, F4, F5, F6, F7, F8 and F9 were separated. Each fraction was tested by ELISA for antigenicity using sera from G. explanatum-infected field buffaloes. Fractions F1 and F2 were highly antigenic, F3, F4, F6 and F7 were moderately antigenic and F5, F8 and F9 were poorly antigenic. Analyses by SDS-PAGE revealed that each fraction comprised several polypeptide(s) in the molecular weight range of <29 to >205 kDa. Results of Western blotting indicated that not all polypeptides which appeared in the SDS-PAGE were antigenic. The antigenic molecules of each fraction were mostly in the low molecular weight range of <14 to >94 kDa with the polypeptides in the range of >14, 14, 18, 21-25 and 34-36 kDa.