Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

Structural characterization of proteoheparan sulfate isolated from plasma membranes of an ascites hepatoma,AH 66

A proteoglycan isolated from plasma membranes of an ascites hepatoma, AH 66, was characterized structurally. The glycosaminoglycan was obtained by alkali treatment and was identified as heparan sulfate. It was essentially the only type of carbohydrate chain attached to the core protein. The identification was based on chemical analysis, electrophoresis, and digestibility with heparitinase from Flavobacterium heparinum. Analysis of neutral sugars of the proteoglycan by mass fragmentography indicated the presence of xylose and galactose which should be involved in the linkage region between a heparan sulfate chain and the core protein. The weight-average molecular weights of the proteoglycan and its heparan sulfate chain were determined to be 71,000 and 21,000, respectively, by meniscus depletion equilibrium centrifugation. The latter value was in good agreement with those obtained by chemical analysis and by gel filtration. From these values for molecular weight and the protein content of the proteoglycan (10.6%), the molecular weight of the core protein was estimated to be 7500. On the basis of these molecular parameters, it was proposed that three heparan sulfate chains on average are linked to the core protein.     

Purification, characterization, and properties of beta-glucosidase enzymes from Sclerotium rolfsii

Four β-glucosidase enzymes were extensively purified from the culture filtrates of Sclerotium rolfsii and some of their physicochemical properties studied. All the enzymes showed a single protein band in sodium dodecyl sulfate-gel electrophoresis and in disc gel electrophoresis at pH 8.9 and 4.3. The purified β-glucosidases were free of endoglucanase (carboxymethyl cellulose viscosity-lowering activity). All the enzymes are glycoproteins and are composed of one polypeptide chain. The molecular weight of the four β-glucosidases varies between 90,000 and 107,000. The pH and temperature optima of the four β-glucosidases are 4.2 and 68 °C with p-nitrophenyl-β-d-glucoside and 4.5 and 65 °C with cellobiose as substrate. The isoelectric points for the enzymes are 4.10, 4.55, 5.10, and 5.55, respectively. The specific activities of the enzymes with cellobiose as substrate are 55, 78, 175, and 51 μmol glucose released per minute per milligram protein, respectively. The enzymes are inhibited by the reaction product glucose, and by glucono-δ-lactone and nojirimycin. A carboxylate group is implicated in the catalysis of β-glucosidase.     

Resealing to small solutes of white erythrocyte membranes after incubation with EDTA, Ca2+, salt, sucrose, phospholipase C

White, stable erythrocyte ghosts can recover their impermeability to small solutes after storage for several days in low-ionic-strength phosphate buffers at 0 °C. The accessibility, to their substrates, of the inner surface enzymes, glyceraldehyde-3-phosphate dehydrogenase, (G3PD), and NADH cytochrome c oxidoreductase, was used to assess resealing. The data from the two enzymes were confirmatory. None of the conditions used to investigate resealing altered the activity of the outer surface enzyme, acetylcholinesterase. Using G3PD activity, ghosts (freshly prepared by gentle stepwise hemolysis in hypotonic phosphate buffers and stored in 11 mm phosphate buffer, pH 7.4) were shown to be slightly sealed (33%). Incubation at 37 °C in the storage buffer with or without EDTA did not alter their permeability. Ionic strength rather than osmotic pressure appears to influence the sealing process since salt (286 mosm) elicited 91% sealing whereas sucrose (278 mosm) had little effect. Calcium in trace amounts caused resealing to 80%. Phospholipase C (C. welchii) completely abolished Ca²⁺-induced resealing. The data were highly reproducible although these ghosts were found to contain only 10 to 20% of the G3PD activity of the leaky ghosts prepared by shock hemolysis in 5 mm phosphate buffer, pH 8.0. The response to the resealing agents was similar regardless of the level of G3PD present. Neither calcium nor ETDA altered the chemical composition (sialic acid, cholesterol, phospholipid) of the membranes. The small amount (5%) of nonspecific loosely bound protein lost during incubation, could not be attributed to any of the test agents. The results suggest that calcium induced the recovery of impermeability by altering the association, distribution, and/or conformation of the proteins and phospholipids within the membrane.     

Purification and characterization of the major sialoglycoproteins of the rat erythrocyte membrane

The major sialoglycoproteins of the rat erythrocyte membrane were purified by hot phenol partitioning followed by cation-exchange chromatography on SP-Sephadex. Further purification was obtained by extraction with n-butanol and anion-exchange chromatography on DEAE-cellulose. The resulting sialoglycoprotein fraction was free of lipids and nonsialylated glycoproteins and gave rise to four major periodic acid-Schiff staining bands when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fastest migrating protein on these gels with an apparent molecular weight of 19,000 was purified to homogeneity by gel filtration. The amino acid and sugar compositions of these materials are reported. The protein moiety is rich in serine, threonine, and hydrophobic amino acids and the carbohydrate moiety is high in sialic acid and N-acetylgalactosamine. Most of the carbohydrate is linked O-glycosidically to serine and threonine residues, as shown by susceptibility to base-catalyzed β-elimination and concomitant reduction of serine and threonine to alanine and α-aminobutyric acid and of N-acetylgalactosamine to N-acetylgalactosaminitol in the presence of reducing agents. The significance of these data in light of the known role of the rat erythrocyte membrane sialoglycoproteins in erythropoiesis is discussed. The properties of the rat erythrocyte membrane sialoglycoproteins are compared to those of other species.     

Multiple forms of rat liver mitochondrial DNA polymerase.

Mitochondrial DNA polymerase (DNA polymerase mt) exists in two active forms. DNA polymerase present in crude extract (M-I) and ammonium sulfate precipitate (M-II) stages of purification sediments at 12.1S. The enzyme at the M-II stage of purification has a molecular weight of approximately 250,000 as determined by Sephadex G-200 chromatography in buffers of low ionic strength. In buffers containing 0.15 m NaCl, the enzyme sediments at 9.4S and has a molecular weight of approximately 190,000. When the enzyme is further purified on diethylaminoethyl cellulose (M-III stage of purification), the 9.4S activity predominates. Addition of a polymerase-free fraction from the M-III stage of purification changes the sedimentation coefficient of the enzyme from 9.4 to 12.1S.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

Ethanol reduces hepatic estrogen-2-hydroxylase activity in the male rat

Brief exposure to intoxicating levels of ethanol in the male rat produced a marked reduction in a major hepatic enzyme responsible for estrogen metabolism (estrogen-2-hydroxylase). After 4 days of ethanol administration the specific activity of this enzyme decreased by 70% and remained decreased for 6 days following alcohol withdrawal. Enzyme activity returned to control levels by two weeks. However, if animals were retreated with ethanol for one day each week the enzyme activity remained low. Kinetic analysis of the enzymatic activity from ethanol-treated rats showed a decrease in specific activity (Vmax) with no alteration in substrate affinity (apparent Km). The decrease in enzyme activity persisted long after ethanol disappeared from the blood and concentrations of ethanol from 20–100 mM had no effect on enzyme activity when added $\text{in vitro}$. A similar effect of ethanol on hepatic estrogen metabolism in humans may partially explain the elevated serum estrogen levels and the signs of hyperestrogenization observed in male alcoholic patients.     

Biochemical and electropharmaceutical studies with tricyclic antidepressants in rat and guinea-pig cerebral cortex.

Chopped guinea pig cerebral cortex was incubated with a series of antidepressant drugs which produced increases in the cyclic AMP content of the tissue. These effects were partially or wholly blocked by theophylline, suggesting that they were mediated by endogenous production, release and action of adenosine. A similar series of drugs was iontophoretically ejected on rat cerebral cortical neurons where augmentation of concurrently ejected adenosine was observed as slowing of the rate of cell firing. Pharmacological correlations between the two sets of data suggest a common mechanism of action.     

Cellular aging studied by the reconstruction of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells

Cytochalasin B (CB) was used to enucleate cells (cytoplasts) and to obtain karyoplasts (nuclei) from the human diploid fetal lung fibroblast strain WI-38. Fusion of cytoplasts and nuclei from young and old cells was accomplished with the aid of inactivated Sendai virus. Viable nuclei may be obtained from the karyoplast pellet after passage through a layer of bovine albumin which retains any contamination cytoplasts. The majority of successful fusions forming “whole cells” occurred when cytoplast from “old” cultures (PDL 40–51) and karyoplasts from “young” cultures were used (PDL 12–22), but almost always resulted in limited division of the viable reconstructed cells. When successful fusion occurred between “young” cytoplasts and “young” karyoplasts the number of cell divisions obtained was comparable to control cells kept under similar conditions.     

Heterogeneity of renin substrate released from hepatocytes and in brain extracts

Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. On polyacrylamide gel electrophoresis (PAGE), the hepatocytes medium and brain extracts contained forms of substrate with reduced mobility as compared to the plasma form. Intraperitoneal injection of 17β estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF or PAGE profiles was evident. There was no remarkable change of substrate concentration in the brain following these treatments. Molecular weights of renin substrate were 60,000–65,000 from all preparations. It remains to be established whether the different forms of renin substrate from hepatocytes represent precursor forms of circulating plasma substrate. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.     

A serotonin sensitive adenylate cyclase in mature rat brain synaptic membranes

Results from this study have indicated serotonin-sensitive adenylate cyclase activity in adult rat brain. The enzyme is localized in the synaptosomal plasma membrane and apparently has multiple activation sites for serotonin with specific activity maxima occuring at serotonin concentrations of 5 × 10^?10, 5 × 10^?9, 1 × 10^?8, and 5 × 10^?8 moles/liter. The production of cyclic AMP at these sites appears to be unaffected by 1 × 10^?5M fluphenazine, while 1 × 10^?5M tryptamine, methysergide, and ergonovine decreased the stimulatory effect of 1 × 10^?8M 5-HT by 30 percent, 80 percent, and 57 percent respectively.     

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O₂^? production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca²⁺ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O₂^? generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca²⁺; (4) The amount of O₂^? produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O₂^? and augmented twice the amount of H₂O₂ recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O₂^? but blocked H₂O₂ release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H₂O₂ was derived by enzymatic dismutation of O₂^?; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.     

Soman and sarin induce a long-lasting naloxone-reversible analgesia in mice

Soman (50 micrograms/kg) and sarin (120 micrograms/kg), potent organophosphate anticholinesterase agents, produced an analgesic response in the mouse hotplate latency test. Naloxone antagonized but did not completely reverse the soman- and sarin-induced analgesia, whereas atropine antagonized completely the soman-and sarin-induced analgesia. Soman poisoning did not potentiate morphine-induced analgesia. It was simply an additive response. In survivors of soman (287 micrograms/kg) poisoning, the analgesia was more pronounced and was still apparent 96 hr after administration. This analgesia was completely antagonized by naloxone. Similar results were found in survivors of sarin (510 micrograms/kg) poisoning. The organophosphate-induced analgesia was not due to physical incapacitation as evidenced by performance on the accelerating rotorod. It is suggested that the organophosphate-induced analgesia is due to a combination of an increased concentration of acetylcholine due to inhibition of acetylcholinesterase combined with a reduced destruction of endogenous opioid-like substances due to organophosphate inhibition of proteases.     

Binding of [125I]Bolton Hunter conjugated eledoisin to rat brain cortex membranes--evidence for two classes of tachykinin receptors in the mammalian central nervous system

[125I]Bolton Hunter conjugated eledoisin was prepared and purified by ion-paired reverse phase chromatography. The ligand binds to rat brain cortex membranes, and the binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). The binding site appears to be distinct from the [125I]Bolton Hunter conjugated substance P receptor based on the relative potencies of substance P, eledoisin, kassinin, physalaemin and [pGlu]substance P (6-11) hexapeptide to displace the binding of these two ligands.     

A cellular activator of catecholamine-sensitive adenylate cyclase in rat reticulocytes and erythrocytes: changes during reticulocyte development and effects on the beta receptor

Rat reticulocytes contain an isoproterenol-sensitive adenylate cyclase activity which is lost with maturation to erythrocytes despite no change in the density of β-adrenergic receptors. To explore this observation, a cytosol factor, previously shown to be important in the expression of catecholamine-sensitive adenylate cyclase in the reticulocyte, was compared to a cytosol factor obtained in a similar manner from mature erythrocytes. The cytosol factor from reticulocytes augmented isoproterenol-responsive adenylate cyclase activity in reticulocyte and erythrocyte membranes half-maximally at 0.7 ± 0.1 (SEM) and 1.1 ± 0.3 μg/ml, respectively. These concentrations of reticulocyte-derived cytosol factor were significantly lower (P < 0.01) than those concentrations of the factor from erythrocytes necessary to augment isoproterenol-responsive adenylate cyclase activity in reticulocyte (9.7 ± 2.3) and erythrocyte (7.5 ± 1.0) membranes. Cytosol factor from reticulocytes also caused greater total isoproterenol responsiveness than that from erythrocytes both in reticulocyte (784 ± 107 vs 525 ± 65 pmol/mg protein) and in erythrocyte membranes (54 ± 6 vs 36 ± 3); P < 0.05. Neither reticulocyte nor erythrocyte cytosol factor affected the concentration at which isoproterenol half-maximally stimulated adenylate cyclase in either set of membranes. However, the cytosol factor from reticulocytes markedly decreased the binding affinity of isoproterenol for β receptors in reticulocytes from 0.8 ± 0.2 to 6.9 ± 1.4 μm; P < 0.001. This reticulocyte factor had no significant effect on the binding affinity of isoproterenol for erythrocyte membranes. Erythrocyte factor did not change the binding affinity for isoproterenol in either reticulocyte or erythrocyte membranes.     

Antidote effect of sodium fluoride against organophosphate poisoning in mice

Pretreatment of mice with atropine (17.4 mg/kg) + NaF (5 or 15 mg/kg) had a significant antidotal effect over atropine alone against the lethality produced by soman and sarin. Atropine + NaF (15 mg/kg) was effective against tabun, whereas the lower dose of NaF was not. An effect of NaF on organophosphate inhibited acetylcholinesterase could not account for the antidotal action of NaF. NaF had no effect on liver somanase activity but inhibited aliesterase activity. Aliesterase activity in NaF pretreated somanpoisoned mice was significantly (p < 0.05) higher than in those receiving atropine alone. In CBDP-pretreated mice NaF did not significantly attenuate the toxicity of soman. It is hypothesized that the antidotal effect of NaF versus organophosphate poisoning is due to its antidesensitizing action at nicotinic receptors in the neuromuscular junction and/or sympathetic ganglia in addition to the proposed increased hydrolysis of sarin and direct detoxification of tabun.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.