The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.     

Myotrophin: purification of a novel peptide from spontaneously hypertensive rat heart that influences myocardial growth

Mechanisms involved in the development or the regression of myocardial hypertrophy cannot be fully explained as responses to blood pressure control alone. We had hypothesized that the development of hypertrophy is initiated by a signal (mechanical or humoral) to the myocardium, which in turn produces a soluble factor that triggers protein synthesis and initiates myocardial growth. Using the stimulation of protein synthesis in isolated cardiac myocytes obtained from normal rat hearts as an assay system, we have identified a soluble factor from the hypertrophied myocardium of spontaneously hypertensive rats. This factor, which has been purified to apparent homogeneity, is a protein of 12 kDa. The sequence of three internally liberated peptides containing 7-24 residues was determined. Based on the determined amino acid sequences of these peptides, this factor (designated myotrophin) appears to be a novel protein that shows no homology with any previously described growth factors. Myotrophin is present in human, dog, and rat hypertrophied hearts (28-35% stimulation of protein synthesis over control) and in small amounts in normal hearts (5-6% stimulation). Myotrophin causes two dose-dependent effects in neonatal cardiac myocytes: an increase in the surface area of the myocyte and the appearance of organized myofibrils, which become apparent within 48 h. Myotrophin may play an important role in the pathogenesis of cardiac hypertrophy as well as in the normal development of cardiac myocytes.     

Rapid purification of enzymes of fatty acid biosynthesis from rat adipose tissue

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protein kinases.     

The extraction and purification of a cysteine transfer ribonucleic acid from baker''s yeast.

1. A modification of the RPC 1 system of A.D. Kelmers, G.D. Novelli & M.P. Stulberg (1965) (J. Biol. Chem. 240, 3979-3983) is described in which the support medium is a Celite of narrow range particle size treated with dichlorodimethylsilane. 2. By using this system an apparently pure preparation of tRNA Cys was isolated from baker's yeast tRNA. 3. This preparation accepted at least 60% of the theoretical quantity of [3-14C]cysteine in a conventional assay and failed to accept isoleucine, phenylalanine, proline, serine or tyrosine. 4. A theoretical countercurrent-distribution curve calculated by assuming a distribution coefficient K of 2.03 was in excellent agreement with the profiles of E260 and cysteine-acceptor ability after 537 transfers in the 1.85 M-phosphate/formamide/propan-2-ol system of C.M. Connelly & B.P. Doctor (1965) (J. Biol. Chem. 241, 715-719). 5. Chromatography of tRNA Cys on Bio-Gel P100 polyacrylamide beads afforded two components one of which was far less efficient than the other in accepting cysteine. The base compositions of the two were similar.     

Production and partial purification of a peptide antibiotic from Staphylococcus epidermidis

When grown on solid or in liquid Brain Heart Infusion at 37°C, Staphylococcus epidermidis NCIB 11536 produced antibiotic activity against a wide range of Gram positive bacteria. Production was influenced by aeration, pH, glucose concentration and specific growth rate. Inhibitory activity could be concentrated by ammonium sulphate precipitation (30–55% saturation). On Sephadex G50 using 0.05 mol/1 sodium phosphate buffer, pH 6.0, two peaks of antibiotic activity were detected. The first peak eluted with the void volume (K_d= 0) and the second peak was retained by the gel (K_d= 0.73–0.77). These two substances did not represent the monomeric and polymeric forms of a staphylococcal bacteriocin. The low mol. wt inhibitor, which was responsible for over 95% of the recovered activity on Sephadex G50, could be partially purified by a combination of gel filtration on Biogel P2 and ion-exchange chromatography on Sephadex C-25. Yields were increased by combining these two steps into a single procedure (duocolumn). The semi-purified inhibitor was desalted using Sep-pak C₁₈ cartridges. Biological activity was resistant to enzymic denaturation except by high concentrations of trypsin (50 units/μg, 3 h, 25°C). This peptide antibiotic is different from any previously described staphylococcal inhibitors.     

Expression and purification of a nanostructure-forming peptide

Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P₁₁-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.     

The purification of lactoferrin from human whey by batch extraction

The isolation of lactoferrin from human whey has been accomplished using a rapid two-step procedure. The lactoferrin is directly adsorbed to cellulose phosphate by batch extraction and eluted by a stepped salt and pH gradient. The major impurity, a low-molecular-weight fraction, is quickly removed by gel filtration. The recovered lactoferrin has a purity of about 96%. The yield of lactoferrin averaged 80%. This method of lactoferrin purification greatly reduces the labor and time required, and the procedure is easily scaled to any volume of starting material.     

Rapid purification of a prekallikrein from rat pancreas

A prekallikrein from rat pancreas was purified 1500-fold with an overall yield of 20% using a rapid, simple procedure. DEAE-Sephadex A-50 chromatography permitted the separation of two prekallikrein components present in rat pancreatic homogenates; the major fraction was further purified by Sephadex G-100 gel filtration and immunoadsorption chromatography. The zymogen is a single-chain molecule with pI 4·35. Apparent M_r values of 38,000 and 37,000 were determined by sodium dodecyl sulfate-polyacrylamide gradient electrophoresis and gel filtration, respectively.     

Affinity purification of fibrinogen using a ligand from a peptide library.

An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.     

The purification of a detergent-soluble glucose-6-phosphatase from rat liver.

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5'-phosphate from the cytoplasmic side.     

5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.

Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.     

The purification and properties of aldose reductase from rat ovary

Aldose reductase has been highly purified from rat ovary to apparent homogeneity, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme proved to be a monomeric protein with a molecular weight of about 39,900. The enzyme catalyzed the NADPH-dependent reduction of a number of aromatic and aliphatic aldehydes as well as aldo-sugars. The enzyme was potently inhibited by p-chloro-mercuribenzoate and a commercially developed aldose reductase inhibitor, M79175. The result of an immunoinhibition study, using antibody against the purified enzyme, indicated that the enzyme was responsible for more than 50% of the overall catalytic activity of D-glucose reduction in rat ovarian cytosol. Western blotting analysis revealed that immunoreactive proteins to anti-ovarian aldose reductase antibody were present in adrenal gland, various reproductive tissues, brain, lung, and heart of rats. Furthermore, ovarian tissues of various species contained immunoreactive proteins, though in small amounts. The enzyme was primarily localized in the granulosa cells and oocytes of all stages of follicular development during the estrous cycle, though it was also found in the corpora lutea cells in the pregnant rats.     

The purification from rat liver of a nuclease hydrolysing ribonucleic acid and deoxyribonucleic acid

1. The purification of a nuclease from rat-liver mitochondria is described. The mitochondria are rendered soluble by treatment with Triton X-100 and, after fractionation with ammonium sulphate and acetone, the active fraction is further purified by chromatography on DEAE-cellulose and Sephadex G-75 to give a purification of over 700-fold. 2. The purified enzyme was only very slightly contaminated with deoxyribonuclease II, phosphodiesterase and phosphomonoesterase. The individual activities of these enzymes did not exceed 0.1% of the activity of the liver nuclease. 3. The purified enzyme attacked RNA more rapidly than denatured DNA and hydrolysed native DNA more slowly than denatured DNA. 4. There is some evidence to suggest that the nucleolytic activity of the purified preparation towards native DNA, denatured DNA and RNA is associated with a single protein. 5. The enzyme is relatively labile but is stabilized in the presence of 20% (w/v) glycerol or 10mm-2-mercaptoethanol.