Equimolar interaction between calmodulin and the Ca2+ ATPase from human erythrocyte membranes

The interaction between calmodulin and the pure, solubilized Ca²⁺ ATPase from human erythrocyte membranes was examined by kinetic titration. The data indicated that the two proteins interacted in a molar ratio of 1:1 with a K_d of 4.2 nm. The dependence of enzyme activity on calmodulin concentration agreed quantitatively with that predicted by kinetic theory.     

A minor sialoglycoprotein of the human erythrocyte membrane

The sialoglycoprotein periodate fuchsin sulfite 2 has about 8% of the sialic acid contained in the sialoglycoproteins of the human erythrocyte membrane. This polypeptide appears to have an apparent monomeric molecular weight of 35,000, somewhat smaller than the monomer of the major sialoglycoprotein (periodate fuchsin sulfite 1) as judged by sodium dodecyl sulfate-polyacry lamide gel electrophoresis, and has frequently been confused with the monomer of the major sialoglycoprotein. Periodate fuchsin sulfite 2 is not labeled by the lactoperoxidase procedure in the intact cell, although it is accessible to neuraminidase and other hydrolases. On the other hand, this component can be labeled by lactoperoxidase on the cytoplasmic surface of open membranes or resealed ghosts. Thus, it is a trans membrane protein. Although most of the other transmembrane proteins of the human erythrocyte membrane are extracted from the membrane by 0.1% Triton X-100 in 7 mm phosphate buffer, pH 7.4, this component is not removed and may be a cytoskeletal component. Trypsin, chymotrypsin, and thermolysin peptides, as well as cyanogen bromide fragments, clearly indicate that the primary sequence of this polypeptide can be distinguished from dimeric or monomeric forms of the major sialoglycoprotein (periodate fuchsin sulfite 1).     

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.     

A fluorescence study of A23187 interaction with phospholipid vesicles

The fluorescence of the ionophore A23187 has been monitored in suspensions of egg yolk phosphatidylcholine (EYPC) and dipalmitoyl phosphatidylcholine (DPPC) vesicles. Both the protonated form of A23187 and the Ca²⁺ complex exhibit fluorescence enhancement when extracted into a hydrophobic environment. Measurements of fluorescence intensity versus lipid concentration were thus used to establish lower limits to the lipid/ water partition coefficients. Values obtained in this way were ? 50 ml water/mg phosphatidylcholine. Quenching of A23187 fluorescence by the spin labels 5NMS (methyl ester of 5-nitroxyl stearate), 12NMS, 16NMS, and TEMPO stearamide in EYPC and DPPC vesicles was also investigated. In EYPC all the labels yielded fairly linear Stern-Volmer plots, with TEMPO stearamide quenching about half as strong as the other probes. Quenching in DPPC was generally much stronger than in EYPC, but 12 NMS and 16NMS gave hyperbolic Stern-Volmer plots, apparently due to clustering of the labels. In all the cases the protonated form of A23187 was quenched approximately twice as efficiently as the Ca²⁺ complex, possibly due to a longer fluorescence lifetime for the former. Calculations based on measured spectral properties were performed which indicate that the Förster transfer mechanism extends the nitroxides' quenching range to ～- 10 Å.     

Detection and characterization of a new beta-conglycinin from soybean seeds

A new protein has been isolated from the reserve proteins of the seeds of soybean (Glycine max) which is particularly deficient in methionine and cysteine. The protein dissociated in sodium dodecyl sulfate into a single polypeptide, M_r 48,000. The amino acid composition, N-terminal leucine and mobility on gel electrophoresis of this polypeptide all were indistinguishable from the β-subunit of β-conglycinin. In its nondissociated form, the protein behaved as a trimer of M_r, 137,000 ± 4000. Its sedimentation coefficient at ionic strength 0.5 was 7.5 S and it possessed antigenic determinants in common with β-conglycinin. This protein therefore has the properties of a new isomer of β-conglycinin—a homogeneous trimer of β subunits.     

Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were isolated from several diploid and tetraploid cultivars of perennial ryegrass (Lolium perenne L.) by three different purification protocols. The apparent K_m values for substrate CO₂ were essentially identical for the fully $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$ activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report (M. K. Garrett, 1978, Nature (London)274, 913–915), these results indicate that increased ploidy level (i.e., nuclear gene dosage) has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/ oxygenase in perennial ryegrass.     

Effects of 2-pyridine aldoxime methochloride on cerebral acetylcholinesterase activity and respiration in cats poisoned with sarin

2-pyridine aldoxime methochloride (2-PAM Cl) has been reported to be only marginally effective in reactivating brain acetylcholinesterase (AChE) in animals poisoned with organophosphorus anticholinesterase compounds. In the present studies, an effort was made to obtain additional information on the effects of 2-PAM Cl on AChE activity of the cerebral cortex and respiration in cats intoxicated intravenously with either 1.8 × LD50 (27 μg/kg) or 3 × LD50 (45 μg/kg) of isopropyl methylphosphonofluoridate (Sarin). Following poisoning by 1.8 × LD50 of Sarin, injection of 15 mg/kg of 2-PAM Cl into the common carotid artery in a volume of 5 ml/kg of saline, pH 7.3, affected AChE activity in the cerebral cortex. The enzymatic levels in the poisoned, oxime-treated cats were significantly higher than those of the poisoned, untreated animals. In other experiments, the time for recovery of respiration from the effects of 3 × LD50 of Sarin was reduced from an average of 147 min in untreated animals to within 2 min after oxime administration. Under the conditions of these experiments, 2-PAM Cl rapidly restored respiration and reactivated some AChE in the cerebral cortex of cats intoxicated with Sarin.     

Initiation of mammalian protein synthesis. I. Purification and characterization of seven initiation factors

We have purified seven protein factors from rabbit reticulocytes, all of which are presumed to be involved in the initiation of mammalian protein synthesis. They are termed eIF-1, eIF-2, eIF-3, eIF4A, eIF-4B, eIF-4C and e-IF-5. The purification from the KCl wash of crude ribosomes involves fractionation with ammonium sulphate, ion-exchange chromatography and separation by size. The operational definition of an initiation factor was its requirement for translation of natural messenger RNA (globin mRNA) in a highly purified and fractionated system using completely defined elongation components, i.e. aminoacyl-tRNA, the two elongation factors EF-1 and EF-2, and GTP. By the same criterion ATP was also shown to be required for initiation. The initiation factors were purified to homogeneity with the exception of eIF-4B, which was 60% to 70% pure. They were characterized physically by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. With the exception of eIF-2 and eIF-3, they consist of single polypeptide chains ranging in molecular weight from 15,000 (eIF-1) to about 160,000 (eIF-5). The factor eIF-2 has three subunits of about 35,000, 50,000 and 55,000 molecular weight. The factor eIF-3 appears to be homogeneous as judged by gel electrophoresis in non-dissociating conditions and sedimentation analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, however, reveals at least nine subunits ranging in molecular weight from about 35,000 to 160,000. Initiation complexes (mRNA · Met-tRNA_f · 80 S ribosome), made in the presence of the seven initiation factors, ATP and GTP were isolated on a sucrose gradient and shown to be fully active in polypeptide chain elongation when supplied with aminoacyl-tRNA, the two elongation factors and GTP.     

Focal elevation of liver microsomal epoxide hydrolase in early preneoplastic stages and its behaviour in the further course of hepatocarcinogenesis

Treatment of rats with N-nitrosomorpholine (NNM) for 7 weeks led to a focal increase in liver microsomal epoxide hydrolase (EH) as early as 2 weeks after withdrawal of the carcinogen. This treatment also leads to hyperplastic nodules and liver tumors, but much later. At the same early time point, ATPase activity was decreased in the same islands. Most of these areas already had increased γ-glutamyltranspeptidase activity. The increase in EH at this early time point was more distinct than the decrease in ATPase which has thus far been considered a suitable marker of the earliest stages in hepatocarcinogenesis. The focal increase in EH was also observed in all benign hepatomas, but not in any of the hepatocellular carcinomas investigated so far.     

Vanadate affects glucose metabolism of human erythrocytes

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons and G. Pons (1982) Biochim. Biophys. Acta705, 238–242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.     

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.     

Ca2+ transport against its electrochemical gradient in cytochrome oxidase vesicles reconstituted with mitochondrial hydrophobic proteins

Cytochrome oxidase vesicles have recently been shown to accumulate Ca²⁺ in an energy-dependent manner. Energization of these vesicles with internally trapped cytochrome c and externally added ascorbate and phenazine methylsulfate generated an internally positive membrane potential and prevented Ca²⁺ influx (R. N. Rosier and T. E. Gunter, 1980, FEBS Lett.109, 99–103). In contradistinction, when cytochrome oxidase vesicles were reconstituted with complex V, a mitochondrial protein fraction containing the uncoupler binding site (Y. Hatefi, D. L. Stiggall, Y. Galante and W. G. Hanstein, 1974, Biochem. Biophys. Res. Commun.61, 313–321), both Ca²⁺ uptake and generation of an internally positive membrane potential were observed. The uptake was specifically dependent on energization of electron transport. Control experiments verified that the energization conditions used produced appropriately oriented membrane potentials. Other partially purified hydrophobic mitochondrial protein complexes were found to be less effective than complex V. The reconstituted system showed cation selectivity since Ca²⁺, Mn²⁺, and Rb⁺ were transported, while Na⁺ was not. Low levels of uncoupler, which did not affect oxidation rates, were found to partially inhibit Ca²⁺ uptake regardless of the membrane potential polarity. Uncoupling levels of uncoupler markedly inhibited Ca²⁺ uptake in internally negative cytochrome oxidase vesicles; however, inhibition in internally positive cytochrome oxidase vesicles was less relative to that at lower levels of uncoupler. The uncoupling combination of nigericin, valinomycin, and K⁺ was inhibitory to uptake regardless of membrane potential polarity. A reconstituted system of oxidative phosphorylation, which contains a hydrophobic protein fraction, energized with cytochrome oxidase similarly accumulated Ca²⁺ despite formation of an internally positive membrane potential. The results suggest that cytochrome oxidase, when coupled to appropriate hydrophobic mitochondrial proteins, can act as an electrogenic Ca²⁺ pump deriving its energy directly from electron transport.     

Purification and immunological characterization of human myocardial MB creatine kinase

Elevated plasma MB creatine kinase (CK) is considered the most sensitive and specific diagnostic indicator of myocardial infarction. However, attempts to purify human MB CK have been unsuccessful. The need for purified human MB CK was further enhanced with the development of a radioimmunoassay for CK isoenzymes which would provide more prompt and specific detection of myocardial infarction. The major protein contaminant of MB CK is albumin which has been difficult to separate due to their similar electrophoretic mobility. Human hearts were obtained within 2 h postmortem and the tissue homogenized in 50 mm Tris-HCl (pH 7.4), 2 mm mercaptoethanol. The CK was recovered from the supernatant (31,000g) by ethanol extraction (50–70%). The resuspended pellet was fractionated on DEAE Sephadex A-50 with a salt gradient (50–500 mm, pH 8.0). The MB fraction contained about 90% albumin. The preparation was bound to an Affigel blue column and contaminating proteins other than albumin were eluted with 50 mm Tris-HCl (pH 8.0), 2 mm mercaptoethanol. MB CK was eluted with 250 mm NaCl, but the albumin remained bound. The MB fraction with a specific activity of 453 IU/mg represented an 80-fold increase in purity and exhibited a single protein band on polyacrylamide gels. Purified MB CK labeled with ¹²⁵I exhibited no binding to human albumin antiserum, but bound to MB CK antiserum, and unlabeled MB CK competitively inhibited binding of ¹²⁵I-MB CK in the radioimmunoassay system exhibiting a sensitivity for detection of plasma MB CK at the nanogram level.     

pp60c-src Kinase is in chick and human embryonic tissues

The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.     

A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: Isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus

A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na⁺ within the vesicles for external Cs⁺. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.     

Purification of galactose-1-phosphate uridylyltransferase from human placenta

Galactose-1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been purified 4000-fold from human placenta in four chromatographic steps using DEAE-cellulose, hydrocylapatite, ethyliminohexylagarose, and Sephacryl S-200. The specific activity of the homogeneous enzyme was 56 units/mg protein. The placental enzyme consists of two similar subunits, each of molecular weight about 48,000. The placental enzyme was similar to published results for the red cell enzyme (V. P. Williams, Arch. Biochem. Biophys., 1978, 191, 182–191) with respect to subunit molecular weight, electrophoretic migration, and immunological properties. The more purified fractions of the placental enzyme invariably contained a glycoprotein which was removed in the gel filtration step. After this glycoprotein was removed, the enzyme was very labile and only about 20% of the catalytic activity was recovered.     

Isolation and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes.

A rapid and convenient procedure for isolating human glyceraldehyde-3-phosphate dehydrogenase from erythrocytes has been developed and yields enzyme with a specific activity of 33–52. The physical and catalytic properties of the enzyme are similar to those of rabbit muscle enzyme. Reassociation of freshly isolated human glyceraldehyde-3-phosphate dehydrogenase with washed erythrocyte membranes increases the specific activity and stability of the enzyme suggesting that enzyme-membrane interactions may have an important effect on the conformation and catalytic activity. That the human enzyme behaves as a dimer of dimers, similar to the behavior or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, is suggested by its half-of-the-sites reactivity toward 4-iodoacetamido-1-naphthol. The human enzyme binds nicotinamide hypoxanthine dinucleotide, a structural analog of NAD⁺, with negative cooperativity, further indicating its similarity to rabbit muscle enzyme.