Isolation and characterization of a small molecular weight protein of linseed meal

A small M_r, protein from linseed meal has been isolated by CM-Sephadex chromatography. The protein was found to be homogeneous by the techniques of gel filtration, polyacrylamide gel electrophoresis and ultracentrifugation. It had S_20,w value of 1.6S. Amino acid composition of the protein revealed a high amount of glutamic acid, cystine, arginine and glycine. The absorption spectrum of the protein consisted of a peak at 280 nm with a shoulder at 290 nm. The fluorescence emission maximum was at 340 nm. The protein contained large amounts of α-helix and β-structure. SDS-PAGE showed the protein to consist of a single polypeptide chain. The M_r estimated by Archibald's method, sedimentation-diffusion method and gel filtration was 17 000,16 000 and 15 000 respectively. Difference spectra studies as a function of pH and temperature showed no variation in the conformation of the protein, probably due to disulphide bridges.     

Three-dimensional reconstruction and averaging of 50 S ribosomal subunits of Escherichia coli from electron micrographs

The thermal stability and melting kinetics of the α-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil α-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 (M_r = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 (M_r = 16,000 to 17,000) and 24,000 to 26,000 (M_r = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The α-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range (τ_f) and the other in the millisecond range (τ_s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast (τ_f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.     

Properties of γ-glutamyl arylamidase activity of the heavy subunit of γ-glutamyl arylamidase from Bacillus sp. strain No. 12

γ-Glutamyl arylamidase of Bacillus sp. strain No. 12, composed of two heavy (M_r 56 000) and two light (M_r 46 000) subunits, was dissociated and inactivated by mild SDS treatment. The activity was restored in the isolated heavy subunit but not in the light subunit when SDS was removed by dialysis. The restored activity of the heavy subunit was similar to that of the native enzyme with regard to substrate specificity and inhibition and activation by α- and γ-glutamyl compounds, free amino acids, peptides, enzyme inhibitors, and anti-native enzyme antibody.     

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (M _r 1000–4000) fragment from the chain resulted in the generation of a M _r 36 000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the M _r 36 000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Enzyme induction in soybean infected by Phytophthora megasperma f.sp. glycinea

Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) M_r on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 M_r whereas plasmin produced two fragments of 180,000 and 140,000 M_r. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 M_r) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule.     

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

The separation and characterization of two forms ofTorpedo electric organ calelectrin

Two methods for extracting calelectrin, a Ca²⁺-regulated membrane-binding protein from the electric organ ofTorpedo marmorata, have been compared and the more promising one was modified to increase the yield to 7–8 mg · kg⁻¹ wet weight of tissue, that is 4–5-times greater than the original method. The calelectrin so obtained could be resolved into a minor component (designated L-calelectrin) eluted from an anion-exchange column at relatively low ionic strength (100 mM NaCl) and a major component (H-calelectrin) eluted at higher ionic strength (300 mM NaCl). The two forms were also separated by chromatography on a hydrophobic resin. Electrophoresis on cellulose acetate indicated that L-calelectrin had a lower mean isoelectric point that the H-form and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed that under reducing conditions (presence of 5% β-mercaptoethanol) both forms migrated as single species, the L-form having a lower apparent relative molecular mass (M_r 32 000) than the H-form (34 000). Under non-reducing conditions, there was no change in the migration of L-calelectrin but the H-form was resolved into two components of M_r 34 000 and 32 000. The addition of 2 mM Ca²⁺ had no effect on the migration of either form. Both forms were equally recognized by an anti-calelectrin antiserum and were microheterogeneous with respect to their isoelectric points (pH 4.3–5.5) in two-dimensional gel electrophoresis. Physical measurements were carried out on the major H-form. The Stokes radius was estimated to be 3 nm, corresponding to an apparent M_r of 44 000. It was unaffected by changes in ionic strength, pH or Ca²⁺ concentration. Analytical ultracentrifugation gave a sedimentation constant of 2.9 S and an apparent M_r of 36 000. Measurements of circular dichroism indicated that 78% of the molecule was in the α-helix configuration and 22% in random coil. Ca²⁺ had no significant effect on the conformation.     

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation

Incubating rat fat cell membranes with [³²P]NAD⁺ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42 000, 46 000 and 48 000. Partial proteolytic peptide maps of the M_r = 46 000 and 48 0000 toxin-specific substrates generated by elastase, α-chymotypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the M_r = 42 000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the M_r = 42 000 target displayed a number of peptides which were absent from the maps generated from either the M_r = 46 000 or 48 000 targets. These data suggest that the M_r = 46 000 and 48 000 substrates are closely related proteins, however the relationship between the M_r = 42 000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [³²P]NAD⁺     

Tandem genetic duplications in Salmonella typhimurium: amplification of the histidine operon.

Bovine pancreatic phospholipase A₂ (M_r = 14,000) has been crystallized and its three-dimensional structure determined by X-ray diffraction analysis to a resolution of 2.4 Å. Three heavy-atom derivatives were used in the phase calculations with inclusion of the anomalous dispersion differences. The resulting electron density map allowed an easy and unambiguous tracing of the peptide chain. Two of the seven disulfide connections appeared to be different from what was suggested by the earlier chemical and structural work. The bovine phospholipase A₂ structure contains about 50% α-helix and 10% β-structure. The bovine enzyme structure was found to deviate substantially from the previously published porcine prophospholipase structure.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

The structure of rooster-comb dermatan sulfate. Fragmentation of the polysaccharide chains by chondroitinase AC-II digestion,and by periodate oxidation,followed by alkali cleavage

The polysaccharide-chain fragments of rooster-comb dermatan sulfates (RC-20 and RC-30) were obtained by chondroitinase AC-II digestion and by periodate oxidation, followed by alkaline cleavage, and their structures analyzed both quantitatively and qualitatively. RC-20 having a lower d-glucuronic acid content (22.6%) is composed preponderantly of large clusters of N-acetyldermosine sulfate (M_r～17 600–41 000) at the nonreducing terminal, whereas RC-30, having a higher d-glucuronic acid content, (41.4%) is poor in this cluster. Both RC-20 and RC-30 have an N-acetyldermosine sulfate cluster (M_r 6500–7300) within the polysaccharide chains. Most N-acetylchondrosine sulfate units of RC-20 and RC-30 exist as clusters, the large clusters (M_r～17 600) being preponderant in RC-30; both RC-20 and RC-30 contain a large proportion of N-acetylchondrosine sulfate clusters (M_r 3500 and 9000) that corresponds to the uronic acid content. In RC-30, most N-acetyldermosine disulfate units (13.4%) are linked to N-acetylchondrosine sulfate units or clusters.     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/μmol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [¹²⁵I]ICYP-diazirine binds with high affinity (K_d = 60 pM) to β-receptors from turkey erythrocyte membranes. Upon irradiation, [¹²⁵I]ICYP-diazirine is covalently incorporated in a M_r 40 000 protein. Stereoselective inhibition of photolabeling by the (?)enantiomers of alprenolol and isoproterenol indicated that the M_r 40 000 protein contains a β-adrenergic binding site. The yield of specific labeling was up to 8.2% of total β-receptor binding sites. The M_r 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [¹²⁵I]ICYP-diazirine resulted in specific labeling of two proteins with M_r 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximate M_r 67 000) was labeled specifically.     

Coupling of the α1-adrenergic receptor to a guanine nucleotide-binding regulatory protein by a discrete domain distinct from its ligand recognition site

At rat hepatic membrane α₁-adrenergic receptors, the nonhydrolyzable GTP analogue p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding. This p[NH]ppG effect is consistent with the involvement of a guanine nucleotide-binding regulatory protein (G-protein) in α₁-adrenergic receptor signalling. Although readily apparent in membranes prepared to avoid retention of endogenous nucleotides and activation of Ca²⁺-sensitive proteinases (+pi), this p[NH]ppG effect is not observed in membranes prepared without proteinase inhibitors (−pi), or in −pi membranes treated with Ca²⁺ (−pi, +Ca²⁺). In these various membrane preparations, different M_r forms of the receptor are also identified by photoaffinity labelling with [¹²⁵I]CP65 526, an aryl azide analog of the α₁-selective antagonist, prazosin, followed by SDS-polyacrylamide gel electrophoresis and autoradiography. Whereas a predominant M_r = 80 000 subunit is identified in +pi membranes, in −pi membranes a proteolytic M_r = 59 000 fragment is also observed. In −pi, +Ca²⁺ membranes, only this latter peptide is detected. To evaluate the ability of each of these forms of the receptor to couple with a G-protein, the effect of p[NH]ppG on the agonist-inhibition of [¹²⁵I]CP65 526 labelling was determined by laser densitometry scanning and computer analysis. At the M_r = 80 000 subunit, p[NH]ppG causes a rightward shift of agonist competition curves and a loss of high-affinity binding, even in −pi membranes. By contrast, agonist-binding at the M_r = 59 000 subunit is of low-affinity and was not affected by p[NH]ppG. These data indicate that the cleaved M_r = 59 000 fragment, while retaining hormone binding activity is unable to undergo G-protein coupling. Thus, the α₁-adrenergic receptor appears to contain a discrete domain necessary for G-protein coupling that is distinct from its ligand recognition site.     

Calmodulin from Pharbitis nil: Purification and Characterization

A protein, identifiable as calmodulin (CaM), has been isolated from the seedling tissue of Pharbitis nil. The method has been developed to isolate a high quality protein from plant tissue containing the high content of polyphenols. This protein was relatively heat-stable and bound to hydrophobic resin in calcium-dependent manner. It was recognized by the antibody against pea and carrot, but did not bind to antibody against Dictyostelium discoideum. This protein had M_r of 15 kDa and 18.5 kDa in the presence and absence of Ca₂₊, respectively, and was able to stimulate calmodulin-deficient cAMP phosphodiesterase. Based on its migration on SDS-PAGE gels, M_r and binding to anti-CaM antibodies it was deduced that calmodulin from P. nil is essentially identical to calmodulin isolated from other plants.     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.