Dependence of aggregation and ligand affinity on the concentration of the folate-binding protein from cow's milk

Ultracentrifugation and gel-filtration studies showed that the folate-binding protein from cow's milk possessed a remarkable aggregation tendency at pH 7.4. Aggregation was enhanced in the presence of folate which suggested an interrelationship between the mechanisms of ligand binding and polymerization. The degree of polymerization increased with increasing concentrations of binding protein. Thus, while the monomer prevailed at 1 nM, a polymer composed of more thn 32 monomers was formed at 130 microM. Two characteristics of folate binding, i.e., Scatchard plots that were convex upward and a ligand affinity that was inversely proportional to the concentration of binding protein, could be interpreted in terms of ligand binding to a polymerizing system in which the polymerization equilibria affect the ligand affinity.     

On the role of serotonin and 5-methoxy-tryptamine in the regulation of cell division in sea urchin eggs

The presence of serotonin in sea urchin eggs has been ascertained by high-performance liquid chromatography and thin-layer chromatography analysis of tissue-free bioamines. The results show the presence of both serotonin and 5-methoxytryptamine. The role of these substances in the cell division process has been studied by using the serotonin antagonists, gramine and metergoline. Both antagonists cause a significant delay of the cell division which, however, can be prevented by the addition of either 5-hydroxytryptophane, serotonin, or 5-methoxytryptamine. The effect of gramine on the different stages of the cell division process has also been investigated. Neither S phase nor mitosis are affected by the serotonin antagonist, while cleavage is delayed. The effect of serotonin seems mediated by calcium ions and cAMP. Gramine causes a marked increase in radio-calcium efflux from the fertilized egg, and at the same time lowers the cAMP level.     

Preparation of plasma membranes from fertilized sea urchin eggs

A new method is presented for preparation of highly purified plasma membranes from fertilized sea urchin eggs. The purified plasma membranes are in vesicle form and are highly enriched in ouabain inhibitable, Na+/K+ ATPase activity. Analysis of membrane proteins by sodium dodecyl sulfate-gel electrophoresis indicates that several high-molecular-weight proteins characteristic of plasma membranes from unfertilized eggs are absent in plasma membranes from fertilized eggs.     

Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes

Enzymes situated along the luminal surface of pulmonary endothelial cells interact with circulating solutes, notably with vasoactive substances, to regulate the hormonal composition of systemic arterial blood. However, it is becoming clear that the range and complexity of reactions occurring at or near the surface of endothelial cells are greater than previously recognized. In addition, evidence indicates that the quality of cell cultures used to define specific endothelial functions must be carefully controlled, together with development of improved understanding of the effects of long-term culture on pulmonary endothelial cells. We have developed new techniques for the culture of pulmonary endothelial cells which avoid exposure to proteolytic enzymes at both the isolation step and during subculture. A combination of mechanical harvest and culture on microcarrier beads has provided a system for the long-term, large-scale culture of pulmonary endothelial cells, features which to a large extent determine the scope of biochemical studies which can be undertaken.     

Purification of glutathione reductase from porcine erythrocytes by the use of affinity chromatography on 2', 5'-ADP-Sepharose 4B and crystallization of the enzyme.

Glutathione reductase has been purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2′,5′-ADP-Sepharose 4B. The enzyme was crystallized from an ammonium sulfate solution. Some of the physical and kinetic properties of the purified enzyme are reported.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Membrane modulation in a methylotrophic bacterium methylococcus capsulatus (Texas) as a function of growth substrate

Methylococcus capsulatus (Texas), when grown on methane, undergoes with age a progressive degeneration of internal membrane structure with a simultaneous accumulation of intracellular inclusions. When M. capsulatus is grown on methanol, virtually no internal membranes are present but, instead, cells contain many intracellular droplets morphologically similar to inclusions in old methane-grown cells. Membranes are regenerated by the cells when a methanol-grown culture is transferred back to methane. The oxidative ability of methane- and methanol-grown cells was compared.     

Alpha 2-macroglobulin binding to cultured fibroblasts: identification by affinity chromatography of high-affinity binding sites

Binding sites having the properties of high-affinity receptors for activated alpha 2-macroglobulin (alpha 2M) have been purified over 100-fold from membranes of spontaneously transformed NIH-3T3 cells (J. A. Hanover, S.-y. Cheng, M. C. Willingham, and I. H. Pastan [1983] J. Biol. Chem. 258, 370-377). To identify the molecular species involved in high-affinity binding, the solubilized receptor has been purified 500-fold by conventional procedures and further purified by affinity chromatography. After radioiodination of the 500-fold-purified preparation, the detergent-solubilized extract was applied to alpha 2M-Sepharose and an 85,000 +/- 5000 Mr species was selectively retained by the column. Binding of the 85,000 +/- 5000 Mr species to the affinity resin was inhibited by EDTA and by excess alpha 2M. Elution from the affinity column could be accomplished with bacitracin, a competitive inhibitor of alpha 2M binding, or with EDTA. Consistent with the previously reported characteristics of the high-affinity alpha 2M receptor, the 85,000 Mr species bound much more efficiently to methylamine-activated alpha 2M-Affigel than to alpha 2M-Affigel which had not been amine-activated. The present data suggest that a protein with a subunit Mr of 85,000 +/- 5000 may represent a component of the high-affinity alpha 2M receptor present on cultured fibroblasts.     

Myocardial infarction in the guinea pig: cellular electrophysiology

The cellular electrophysiology of left ventricular preparations from guinea pig hearts was studied 1 hour, 24 hours, and 4-6 weeks after myocardial infarction produced by 6-8 single ties of the distal left coronary artery system or after sham operation. Microelectrode recordings were used to monitor cells from the endocardial surface of each preparation in tissue bath. All coronary ligated preparations displayed accelerated spontaneous activity compared to normal and sham operated preparations. Single and multiple premature ventricular depolarizations occurred frequently in coronary ligated and rarely in normal and sham operated preparations. Premature stimuli delivered to areas overlying and bordering the area of infarction, induced short bursts of self-terminating rapid repetitive ventricular activity in 4 of 8 (50%) acute (1-hour), 5 of 9 (55%) subacute (24-hour), and 14 of 20 (70%) healed (4-6-week) infarcted preparations. Such activity could not be induced in normal and sham operated preparations. The preparations with healed infarction were unique in that they demonstrated runs of self-terminating repetitive ventricular activity which occurred spontaneously or was inducible with premature stimulation. Recordings from multiple sites in acute, subacute, and healed preparations revealed a variety of transmembrane action potential abnormalities (i.e., reduced action potential amplitude and resting potential, decreased and increased action potential duration, and depressed maximum rates of phase 0 depolarization) in cells overlying and bordering areas of infarction. Only Purkinje fiber action potentials were recorded over the healed infarcts. These data demonstrate that a spectrum of electrophysiological alterations occur in response to ischemic injury and persist after healing of the injury in this new model of myocardial infarction utilizing the guinea pig.     

The structure of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone, an effective analog of colchicine

-Methoxy-5-(2',3',4'-trimethoxyphenyl) tropone is an active analog of colchicine, a mitotic spindle inhibitor, which is missing the middle "B" ring. This compound crystallizes in the triclinic system, space group P1, with Z = 2; a = 10.135(2), b = 10.166 (4), and c = 7.863(2) A; alpha = 82.15(3), beta = 103.49(3), and gamma = 107.16(2); degrees and V = 750.7(4) A. The structure was solved by direct methods and refined by full-matrix least-squares to a final R = 0.063, using 2503 observed reflections and 271 parameters. Despite the absence of the middle ring, the conformation of the molecule is similar to that of colchicine, isocolchicine , and their derivatives. The troponoid ring is dissimilar to the phenyl ring in that it is not aromatic and does have alternating short and long bond lengths. The dihedral angle between the least-squares planes of the two rings is -57.4 degrees. Van der Waals surface representations of the analog and colchicine are presented to demonstrate the similarity and differences of these two molecules . The structural information of the analog is consistent with the interpretation of thermodynamic parameters which govern the interactions between brain tubulin and the analog.     

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: isolation by affinity chromatography and association with the enzymes

Five isoinhibitors, proteins that inactivate chymotrypsin and elastase, were isolated from aqueous extracts of the intestinal parasite Ascaris lumbricoides var. suum by affinity chromatography. They were named in the order that they eluted from a CM-Sephadex C-25 column at pH 8.6 using a salt gradient. Isoinhibitor 1, first reported in this paper, is anionic on polyacrylamide gel electrophoresis at pH 9.3. The other four isoinhibitors are cationic on electrophoresis at pH 9.3, separable from each other, and identical with those reported previously [R.J. Peanasky and G. M. Abu-Erreish (1971) in Proceedings International Research Conference on Proteinase Inhibitors (Fritz, H., and Tschesche, H., eds.), pp. 281-293, de Gruyter, New York]. Amino acid compositions show differences between the isoinhibitors. Antibody to isoinhibitor 1 reacts with its self-antigen only. Antibody to isoinhibitor 5 reacts with isoinhibitors 2-5 but not with isoinhibitor 1. Association equilibrium constants show that each of the isoinhibitors interacts most avidly with alpha-chymotrypsin. For isoinhibitor 1, the K alpha for alpha-chymotrypsin was 2.6 X 10(11) M-1, for porcine elastase I 1.6 X 10(10) M-1, and for Subtilisin Carlsberg 3.3 X 10(7) M-1. For isoinhibitors 2-5, the K alpha ranges were 7.1 X 10(10) to 1.3 X 10(11) M-1 for alpha-chymotrypsin, 1.0 X 10(9) to 5.6 X 10(9) M-1 for porcine elastase I, and 6.0 X 10(8) to 1.3 X 10(9) M-1 for subtilisin Carlsberg. Because of the strong affinity of these inhibitors for alpha-chymotrypsin and elastase, two proteins in the normal environment of the nematode, the name isoinhibitors of chymotrypsin/elastase is suggested for these proteins.     

DEAE paper chromatography to separate intermediates of the pyrimidine biosynthetic pathway and to assay asparatate transcarbamylase and dihydroorotase activities

A DEAE paper chromatographic method was developed to separate the six sequential intermediates on the pyrimidine biosynthetic pathway. This method has been utilized to assay aspartate transcarbamylase and dihydroorotase activities. Consequently, it can be inferred that the remaining three enzymes unique to pyrimidine biosynthesis can also be assayed in a similar manner.     

Interaction of gene 5 protein with DNA

Gene 5 protein bound to both linear and circular single-stranded DNA and saturated the DNA at a protein-to-DNA weight ratio of 7–8. The viscosity of a complex of the protein with single-stranded DNA was initially less than that of the DNA and slowly increased with time suggesting that the complex adopts its final hydrodynamic shape very slowly. This shape change was confirmed by gradient centrifugation. The complex has a more extended structure than DNA alone accounting for its high viscosity and low S value. Gene 5 protein also bound to linear double-stranded DNA though not as strongly as to single-stranded DNA. The protein decreased the transition temperature, T_m, for viscosity loss of double-stranded DNA by 20 °C in 1 and 10 mm salt at a protein-to-DNA ratio of 2.2. At these low ratios there was no decrease in the hyperchromic T_m at 260 nm. At higher ratios of protein to DNA, the hyperchromic T_m was decreased to a constant value and not by a constant amount. Under no conditions was gene 5 protein able to completely separate the complementary strands of double-stranded DNA or to renature denatured DNA.     

Fatty acid desaturase system of yeast microsomes. Involvement of cytochrome b5-containing electron-transport chain.

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 mm. The rate of reoxidation of cytochrome b₅ in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b₅ reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b₅ was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b₅ as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.     

Purification of ferredoxin-NADP+ oxidoreductase from cyanobacteria by affinity chromatography on 2′,5′-ADP-Sepharose 4B

A simple and rapid method for the purification to homogeneity of ferredoxin-NADP⁺ oxidoreductase (EC 1.18.1.2) from the nitrogen-fixing filamentous cyanobacterium Anabaena sp. strain 7119 is described. A crude extract prepared by solubilizing the cells with a detergent was first partially purified on a DEAE-cellulose column and then chromatographed on 2′,5′-ADP-Sepharose 4B. Ligand-bound ferredoxin-NADP⁺ oxidoreductase was eluted by a linear gradient of NaCl. The overall procedure provided an enzyme purified about 400-fold with a yield of 60 to 70%. The final enzyme preparation exhibited a specific activity of 120 units/mg protein and an absorbance ratio $\text{A}{}_{280}\text{A}{}_{458}$ of 8.26. The enzyme protein migrated as a single band when subjected to polyacrylamide gel electrophoresis and chromatographed as a single isoelectric species under chromatofocusing.     

Purification and properties of glycogen phosphorylase a from the muscle of the blue crab, Callinectes danae

Blue crab muscle (Callinectes danae) glycogen phosphorylase a was purified by adsorption of a crude extract on a starch column, elution with a dilute glycogen solution, selective precipitation with ammonium sulfate, dialysis against a solution containing ammonium sulfate and ethylenediaminetetraacetate, followed by centrifugation and chromatography on Sephadex G-25 (sp act 64.5 IU, recovery of 53.8%, and a purification factor of 189). The lyophilized preparation is stable for several months. Disc electrophoresis of the purified phosphorylase yields two protein bands, both with enzymatic activity of the a form. One of the protein bands represents about 10% of the total amount of protein present in the two bands. The molecular weight of the enzyme is 176,000 as determined by ultracentrifugation in a sucrose density gradient and 180,000 as determined by discontinuous polyacrylamide gel electrophoresis. The molecular weight found by disc electrophoresis corresponds to the main protein band. Crab muscle phosphorylase a is not associated under electrophoretic conditions in which rabbit muscle phosphorylase a shows association behavior. Subunit studies by continuous SDS-gel electrophoresis suggest that crab muscle phosphorylase a possesses only one subunit. Pyridoxal-5′-phosphate is a cofactor of the enzyme.     

Anion binding to oxidized type 2 depleted and native laccase: a spectroscopically effective model for exogenous ligand binding to the type 3--type 2 active site

Xanthine oxidase, a mammalian nitroreductase, catalyzed the binding of [³H]1-nitropyrene to DNA. The binding was dependent on the presence of hypoxanthine and was inhibited by allopurinol, a specific xanthine oxidase inhibitor. These data support the hypothesis that nitroreduction is a necessary step in the metabolic activation of 1-nitropyrene to a bacterial mutagen.     

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.