Simultaneous isolation of insulin-like growth factors I and II from adult sheep serum

Ovine insulin-like growth factors I and II (oIGF-I and oIGF-II) have been purified from adult sheep serum. oIGF-II-like receptor-binding activity and IGF-I-like immunoactivity were enriched on SP-Sephadex C-25, then purified using HPLC in the presence of a variety of counter ions. IGF-I- and IGF-II-like activities were separated using HPLC in the presence of 0.2% tetrabutylammonium phosphate at pH 7.0. The final recovery of oIGF-I was 82.6 micrograms from 3.2 litres of adult sheep serum (a yield of 17.6%), and the recovery of oIGF-II was 388 micrograms (a yield of 13.3%). Both IGF preparations were considered to be homogeneous as judged by single sharp peaks during analytical HPLC, and unique N-terminal amino acid sequences. Purified ovine IGFs had molecular weights similar to that of other IGFs (approximately 7000), and the first 30 N-terminal amino acids of both peptides were identical to their human counterparts. The isoelectric points of oIGF-I (pI approximately 8.2) and oIGF-II (pI approximately 6.8) were similar to those of human (h) IGFs (hIGF-I pI approximately 8.2; hIGF-II pI approximately 6.5), and the overall amino acid content of the ovine IGFs was also similar to that of IGFs from other species. oIGF-II preparations from fetal sheep and from adult sheep appeared to be identical. The isolation procedure represents one of general utility that can be easily modified to facilitate the isolation of recombinant IGFs from culture fluid.     

Biochemical characterization of the lizard toxin gilatoxin

The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.     

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26~21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26~21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26~21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).     

Disulfide-linked cyanogen bromide peptides of bovine fibrinogen. I. Isolation of peptide F-CB3 and characterization of its single disulfide bond by cleavage with cyanide.

A fragment F-CB3 which originates from the alpha-chain constituent of bovine fibrinogen could be liberated by CNBr cleavage and was purified by molecular sieve and ion-exchange chromatography. This fragment had a molecular weight of 36 000 and consisted of a single polypeptide chain which is folded into a loop by a single disulfide bridge. Further cleavage of F-CB3 by cyanide or by 2-nitro-5-thiocyanobenzoic acid gave rise to three fragments, CN1, CN2 and CN3, with molecular weights of 23 000, 8000 and 7000, respectively. With both reagents the yield of cleavage did not exceed 50%. Radioactive labeling and amino acid analysis of the purified fragments indicated the order CN1-CN2-CN3 in intact F-CB3. A shorter and apparently degraded form of F-CB3 was observed in some fibrinogen preparations. The shortening involved a region of about 3000 daltons at the N-terminal site of F-CB3, i.e. in fragment CN1.     

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.     

Separation of Component-Polypeptides of Glutenin by Isoelectric Focusing

1. By isoelectric focusing S-cyanoethyl glutenin was observed to be composed of various component-polypeptides having a pI spectrum in a pH range from 6 to 9.

2. During isoelectric focusing a precipitation zone was built up in the column in spite of the presence of 6 m urea. The amount of the precipitate formed was less with S-cyanoethyl glutenin than with S-sulfo glutenin.

3. S-Cyanoethyl glutenin was divided into eight fractions by isoelectric focusing. By starch-gel electrophoresis it was suggested that Fractions I, III and P were mainly composed of a single component.

4. Major N-terminal amino acids of Fractions I, III and P were phenylalanine, glycine and alanine, respectively. In the amino acid composition, distinct differences were observed in the respective fractions, especially in Fraction P. Fraction P showed a much higher content of basic amino acids and a lower content of glutamic acid in comparison with the other two.     

Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts]

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.     

Chemical and physical properties of the carboxypeptidase Y-inhibitor from Baker's yeast.

The purification as well as some characteristics of the carboxypeptidase Y-inhibitor from baker's yeast have been described in a previous report (Matern, H., Hoffmann, M. and Holzer, H. (1974) Proc. Natl. Acad. Sci. U.S. 71, 4874-4878). In this paper, chemical and physical properties of the purified inhibitor are presented. The molecular weight was estimated at 23 400--24 000 and appears to be a monomeric unit. Amino acid analysis and carbohydrate studies are given, showing the existence of three disulfide bonds and one sulfhydryl group per molecule and the absence of carbohydrate residues. The N-terminal amino acid is blocked by an acetyl group. The C-terminal amino acid is lysine. The isoelectric point (pI) is 6.6 and the inhibitor-enzyme complex is stable (at 25 degrees C) betwen pH 5 and 9. The apparent Ki value was calculated as 2.5.10(-9)M.     

Isolation of minax toxins from the venom of the scorpion Buthus minax and their metabolic effects

Two neurotoxins, minax toxins 1 and 2, were isolated from venom of the scorpion Buthus minax from the Sudan. Molecular weights of 7000 and 6800 and 66 and 62 amino acids were found for minax toxins 1 and 2, respectively. Both toxins contain four disulfide bonds, 1 mol each of phenylalanine, histidine, and tryptophan, no free sulfhydryl groups, and no methionine. Both minax toxins 1 and 2 are basic polypeptides with isoelectric points of 8.2 and 9.0, respectively. There is a significant increase in the calcium content of rat hearts envenomated with minax toxins 1 and 2 or crude venom. This confirms earlier electron microscopic findings of calcium deposits in the heart following scorpion envenomation. There is a concomitant decrease in the calcium and phosphorus content of rat serum following envenomation. It seems that neither scorpion toxins nor scorpion venoms affect the mineral metabolism of the bone. The present investigation indicates that scorpion toxins have not only a neurotoxic action but also broader biological effects such as mineral metabolism.     

Purification and some characteristics of the coagulation factor IX from human plasma.

Non-activated coagulation factor IX was purified approx. 10,000-fold from human plasma. The final product was electrophoretically homogeneous and comprised a tingle polypeptide chain with a molecular weight of about 70,000 and a pI of 4.3-4.45. The N-terminal amino acid was glycine. The amino acid and the carbohydrate contents were analysed and a monospecific antiserum to the factor was raised in rabbits.     

Isolation and characterization of glycogen branching enzyme from rabbit liver

Glycogen branching enzyme was isolated from rabbit liver. The highly purified enzyme shows a monomer molecular weight of 71 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and apparent molecular weights of 93 000 by sucrose density gradient sedimentation and 52 000 by gel-exclusion chromatography on Sephacryl S-300. No glucosamine, mannosamine, galactosamine, or sialic acid was detected in the protein. An amino acid analysis is reported. The spectrum of branching enzyme is that of a simple polypeptide, with A1%280nm = 24.6. Highly purified branching enzyme consists of several closely related active enzyme forms that can be resolved by isoelectric focusing in polyacrylamide gel. The major species of pI 5.7 is flanked by less abundant forms of pI 5.6 and 5.8. Seemingly identical enzyme forms are observed in crude extracts of rabbit liver, skeletal muscle, brain, and heart, although the absolute and relative concentrations vary among the tissues. Branching enzyme apparently does not exhibit tissue-specific isoenzymes.     

Purification and properties of staphylocoagulase.

Staphylocoagulase, an exoprotein of coagulase-positive Staphylococci, has been purified to a state in which only trace amounts of contaminating proteins are detectable. Aspartic acid was found as a single N-terminal amino acid in this preparation. The molecular weight is 61 000; the isoelectric point lies as pH 4.53. The amino acid composition was determined.     

A new class of N-hydroxycinnamoyltransferases. Purification,cloning, and expression of a barley agmatine coumaroyltransferase (EC 2.3.1.64)

Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L.) seedlings. The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity for p-coumaroyl-CoA among various acyl donors with a specific activity of 29.7 nanokatal x mg(-1) protein. Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5.20 as determined by isoelectric focusing. The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues. The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT. Recombinant ACT produced and affinity-purified from Escherichia coli had a specific activity of 189 nanokatal x mg(-1) protein, thus confirming the identity of the purified native protein. A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT. Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily. The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis.     

Expression of variant forms of proopiomelanocortin, the common precursor to corticotropin and beta-lipotropin in the rat pars intermedia

Proopiomelanocortin, the common glycoprotein precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), is the most abundant protein synthesized in rat neurointermediate lobes. It represents 30% of the total amount of radioactive proteins obtained after a 1-h pulse incubation with [3H]phenylalanine. Several forms of this protein can be separated by a high-resolution two-dimensional gel electrophoresis technique. The three most abundant species which can be reproducibly characterized by their apparent molecular weights (Mr) and isoelectric points (pI) were called form I (Mr 34 000; pI 8.2), form II (Mr 36 000; pI 8.2), and form III (Mr 35 000; pI 7.3). Additional minor forms, representing together approximately 30% of the total forms I, II, and III combined, are also observed. They have very close molecular weights but differ by their isoelectric points. When glycosylation is prevented by tunicamycin, forms I and II are replaced by a new molecule with the same pI of 8.2 but a slightly lower Mr (32 000). This form is referred to as form T1. Similarly, form III is replaced by form T2 (Mr 33 000; pI 7.3). Forms T1 and T2 are supposed to be nonglycoslyated peptides. They were further characterized by microsequencing and peptide mapping. They both have the same N-terminal amino acid sequence with leucine residues in positions 3 and 11, and they both contain identical [3H]phenylalanine-labeled tryptic fragments, two of them corresponding to the sequences 1-8 of ACTH and 61-69 of beta-LPH. However, a limited digestion with the Staphylococcus aureus (V8 strain) protease generates a collection of peptides different for each form. These results suggest the presence of at least two different gene products corresponding to the major forms of proopiomelanocortin in the rat pars intermedia.     

Isolation of palmitoyl-CoA hydrolases from human blood platelets.

The palmitoyl-CoA hydrolase activity, which in human blood platelets is mainly localized in the cytosol fraction [Berge, Vollset & Farstad (1980) Scand. J. Clin. Lab. Invest. 40, 271--279], was found to be extremely labile. Inclusion of glycerol or palmitoyl-CoA stabilized the activity during preparation. Gel-filtration studies revealed multiple forms of the enzyme with molecular weights corresponding to about 70 000, 40 000 and 24 000. The relative recovery of the mol.wt.-70 000 form was increased by the presence of 20% (v/v) glycerol or 10 microM-palmitoyl-CoA. The three enzyme forms are probably unrelated, since they were not interconvertible. The three different species of palmitoyl-CoA hydrolase were purified by DEAE-cellulose and hydroxyapatite chromatography, isoelectric focusing and high-pressure liquid chromatography (h.p.l.c.) to apparent homogeneity. The three enzymes had isoelectric points (pI) of 7.0, 6.1 and 4.9. The corresponding molecular weights were 27 000--33 000, 66 000--72 000 and 45 000--49 000, calculated from h.p.l.c. and Ultrogel AcA-44 chromatography. The apparently purified enzymes were unstable, as most of the activity was lost during purification. The enzyme with an apparent molecular weight of 45 000--49 000 was split into fractions with molecular weights of less than 10 000 by re-chromatography on h.p.l.c. concomitantly with a loss of activity. The stimulation of the activity by the presence of serum albumin seems to depend on the availability of palmitoyl-CoA, as has been reported for other palmitoyl-CoA hydrolases. [Berge & Farstad (1979) Eur. J. Biochem. 96, 393--401].     

Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.     

Properties of the F0F1 ATPase complex from Rhodospirillum rubrum chromatophores, solubilized by Triton X-100.

1. A cold-stable oligomycin-sensitive F0F1 ATPase complex from chromatophores of Rhodospirillum rubrum FR 1 was solubilized by Triton X-100 and purified by gel filtration. 2. The F0F1 complex is resolved by sodium dodecyl sulfate electrophoresis into 14 polypeptides with approximate molecular weights in the range of 58000--6800; five of these polypeptides are derived from the F1 moiety of the complex which carries the catalytic centers of the enzyme. 3. The purified F0F1 complex is homogeneous according to analytical ultracentrifugation and isoelectric focusing. 4. The molecular weight as determined by gel filtration is about 480 000 +/- 30 000. S020,w is 1.45 +/- 0.1 S and the pI is 5.4. 5. The amino acid composition of the F0F1 complex is compared with the data obtained for the F1 moiety of the enzyme. 6. Quantitative data on the sensitivity to N,N'-dicyclohexyl-carbodiimide as well as kinetic parameters, regarding substrate specificity and dependence of ATPase activity on divalent cations, are reported.     

Insulin-like growth factor I/somatomedin-C: a rapid isolation procedure with FPLC

Insulin-like growth factor I (IGF I)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000-10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS polyacrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pI of 8.3 +/- 0.1. SDS polyacrylamide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measuring the (3H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.