Showing papers in "Journal of Biochemistry in 1983"
TL;DR: A serum lectin specific for mannose and N-acetylglucosamine residues was isolated from human serum to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan and a distinct binding activity toward mannan which did not require Ca2+ was attributed to immunoglobulins (IgG).
Abstract: A serum lectin specific for mannose and N-acetylglucosamine residues was isolated from human serum to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan. The lectin, called mannan-binding protein, was a glycine-rich protein with an apparent molecular size of approximately 600,000 daltons, and had a subunit structure consisting of a single component with an apparent molecular weight of 31,000. Binding of the isolated lectin to 125I-labeled mannan was dependent upon the presence of Ca2+, proportional to the protein added, and a reversible and saturable process. Scatchard plot analysis of binding data indicated the presence of a binding site with a dissociation constant of 2.3 X 10(-9) M and a maximum capacity of 4.3 pmol of 125I-labeled mannan per microgram of protein (2.6 mol of mannan per mol of the protein). The mannan-binding protein, is different from C-reactive protein (CRP) and amyloid P-component (SAP), both of which are serum components known to bind polysaccharides in the presence of Ca2+. A distinct binding activity toward mannan which did not require Ca2+ was attributed to immunoglobulins (IgG).
226 citations
TL;DR: The results provide direct evidence for the participation of gangliosides in such functions as morphological and biochemical differentiation of neuronal cell cultures and the replacement of the effector function not only qualitatively but also quantitatively.
Abstract: To clarify the role of gangliosides in the morphological and biochemical differentiation of neuronal cell cultures, the model cell culture system represented by two neuroblastoma cell lines, GOTO and NB-1, which were established from adrenal gland and metastatic neck lymph node, respectively, was examined. We found that the total ganglioside fraction from human brain had two remarkable effects on these cell lines, which are similar to those of nerve growth factor (NGF): (a) an increase in the cell number, and (b) an increase in the neurite number and the total length of neurites. In these cases, the genuine effector in total gangliosides could not be ascribed to a possibly contaminating NGF-like protein, but rather to a particular molecular species of the gangliosides, GQ1b, which could completely replace the effector function not only qualitatively but also quantitatively. Our results provide direct evidence for the participation of gangliosides in such functions.
224 citations
TL;DR: The results suggest that proliferation of mature rat hepatocytes is regulated by the cell density, and the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.
Abstract: Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.
186 citations
TL;DR: The similarity in their stabilizing effects on collagen and globular proteins suggests that the stabilization mechanism proposed for globular protein can be essentially extended to fibrous proteins: such protein stabilization would be dominantly mediated through a preferential hydration of protein, originating in the water-structure-making character of sugars and polyols.
Abstract: The effects of sugars and polyols on the thermal denaturation temperature, Tm, of acid-soluble collagen from calf skin were studied at pH 4.0 under atmospheric pressure as well as high pressures up to 4,000 atm. Addition of these compounds invariably raised Tm with increases in their concentration over the whole range of pressure. The extent of stabilization by different sugars and polyols is discussed in terms of their different influences on the structure of water. The hydroxymethyl chain length of polyols and equatorial OH groups of the sugars were found to be decisive factors for their stabilizing effect on collagen structure. The similarity in their stabilizing effects on collagen and globular proteins suggests that our stabilization mechanism proposed for globular proteins can be essentially extended to fibrous proteins: such protein stabilization would be dominantly mediated through a preferential hydration of protein, originating in the water-structure-making character of sugars and polyols.
139 citations
TL;DR: Computer-aided analyses were made of the complete amino-acid sequences of two P-450 species, the phenobarbital-inducible major P- 450 of rat liver microsomes (P-450PB) and camphor-hydroxylating P-550 of Pseudomonas putida (P -450cam), and two highly homologous regions were found, HR1 and HR2.
Abstract: Computer-aided analyses were made of the complete amino-acid sequences of two P-450 species, the phenobarbital-inducible major P-450 of rat liver microsomes (P-450PB) and camphor-hydroxylating P-450 of Pseudomonas putida (P-450cam). Statistically significant homology was recognized between the two P-450 sequences, but these sequences were not related to those of other groups of hemoproteins, such as hemoglobins, peroxidases, and cytochrome c's and b's. Two highly homologous regions, HR1 and HR2, and two other weakly homologous regions were found on optimally matched alignment of the P-450 sequences. The secondary structures of the two P-450's predicted by current prediction methods bear strong resemblance at these homologous regions. Both HR1 and HR2 contain a cysteine residue near the center of the homologous regions, and they are the only regions that show significant homology among all 48 combinations of local sequences around the cysteine residues (six on P-450PB and eight on P-450cam). HR1 is located in the N-proximal half of the molecule, is rich in hydrophilic residues, and is predicted to be helical. On the other hand, HR2 is close to the C-terminus, has intermediate hydrophobicity, and may take a complex secondary structure of a turn-sheet-helix. The amino-acid sequences around the HR1 and HR2 regions are also well conserved in another P-450 species, rabbit P-450LM2.
139 citations
TL;DR: Urinary pyridinoline may serve as a useful marker for the breakdown of collagen fibers of skeletal tissues and was increased significantly in patients with certain bone and joint diseases.
Abstract: Pyridinoline, a cross-linking compound of collagen fibers, was found in human urine. A significant portion of urinary pyridinoline was in free form. The ratio of total pyridinoline to creatinine changed with age. It was high in children and decreased with growth. It was low and constant in adults, and increased slightly in old age. It was increased significantly in patients with certain bone and joint diseases. Urinary pyridinoline may serve as a useful marker for the breakdown of collagen fibers of skeletal tissues.
135 citations
TL;DR: Among the various reducing agents tested, ascorbate was most effective in causing DNA scission and protein cleavage, corroborating the possible role of H2O2 in the cleavage reactions.
Abstract: L-Ascorbic acid, when combined with either copper(II) ion or a copper(II)-tripeptide complex, extensively cleaved several viral DNAs and proteins under in vitro conditions. Neither ascorbate nor copper tripeptide (Cu2+-diglycyl-L-histidine) alone caused any apparent changes on these molecules. Various transition metal ions and reducing agents were examined under comparable conditions to determine the basic requirements for both DNA degradation and protein scission activities. Copper and iron are the two most effective transition metal ions examined that exhibit these activities in the presence of ascorbate. The addition of catalase, but not superoxide dismutase, can partially inhibit the scission of DNA in vitro, suggesting that H2O2 may be involved in these activities. Among the various reducing agents tested, ascorbate was most effective in causing DNA scission and protein cleavage, corroborating the possible role of H2O2 in the cleavage reactions. One of the products of the reactions of copper/ascorbate is probably the hydroxyl radical generated from H2O2, which can be formed from the oxidation of ascorbate.
130 citations
TL;DR: To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution.
Abstract: To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Huckel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C.
125 citations
TL;DR: The activities of carnitine octanoyltransferase (COT) and carnitines of saturated fatty acids in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate, and both COT and CPT exhibited catalytic activity with medium- and long-chain acyl derivatives.
Abstract: The activities of carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT) in rat liver were markedly increased by administration of di(2-ethyl-hexyl)phthalate. COT and CPT were purified from the enzyme-induced rat liver. COT was a 66,000-dalton polypeptide. The molecular weight of native CPT was 280,000--320,000 daltons, and the enzyme consisted of 69,200-dalton polypeptides. CAT, COT, and CPT were immunologically different. COT exhibited activity with all of the substrates tested (acyl-CoA's and acylcarnitines of saturated fatty acids having carbon chain lengths of C2--C20), though maximum activity was observed with hexanoyl derivatives. CPT exhibited catalytic activity with medium- and long-chain acyl derivatives. 2-Bromo-palmitoyl-CoA inactivated COT but not CPT. Malonyl-CoA inhibited CPT but not COT. CPT was confined to mitochondria, whereas COT was found in peroxisomes and the soluble compartment but not in mitochondria.
124 citations
TL;DR: A new method for purification of anti-glycosphingolipid antibodies had been developed and it was found that N-Glycolylneuraminyl(alpha 2-3)lactosylceramide [hematoside (NeuGc] could be hydrophobically bound on octyl-Sepharose 4B in the presence of 0.1 M KCl.
Abstract: A new method for purification of anti-glycosphingolipid antibodies had been developed. N-Glycolylneuraminyl(alpha 2-3)lactosylceramide [hematoside (NeuGc)] could be hydrophobically bound on octyl-Sepharose 4B in the presence of 0.1 M KCl. The Sepharose gel coated with hematoside (NeuGc) was used as immunoadsorbent for affinity column chromatography to purify avian anti-hematoside (NeuGc) antibody. The procedure is very simple, reproducible and applicable to purification of almost all anti-glycosphingolipid antibodies. The glycosphingolipid used for the affinity chromatography could be recovered without any destruction by successive extraction of the gel with methanol and methanol/chloroform (1:2, v/v).
115 citations
TL;DR: The 35 K protein was finally identified as glyceraldehyde 3-phosphate dehydrogenase from properties such as mobility on SDS-polyacrylamide gel electrophoresis, cleavage pattern on limited proteolysis, ability to bind to tubulin, and so on.
Abstract: A protein which binds to both tubulin and tubulin polymer was isolated from porcine brains. This protein has a molecular weight of 35,000 on SDS-polyacrylamide gel electrophoresis (designated as 35 K protein). The 35 K protein was purified through several steps of purification including ammonium sulfate fractionation, Sephadex G-100 gel filtration column chromatography, microtubule protein-agarose gel affinity column chromatography and phosphocellulose column chromatography. The 35 K protein caused pronounced enhancement of the turbidity increase produced by tubulin polymerization in the presence of DMSO, but did not have the ability to initiate polymerization of pure tubulin in the absence of DMSO. It was demonstrated that 35 K protein co-sediments with tubulin polymer in a concentration-dependent manner. Electron microscopic observation revealed the formation of bundles of tubulin polymer. Since the effect of 35 K protein was coupled with tubulin polymerization, 35 K protein did not cause the turbidity increase under conditions where tubulin polymerization was inhibited by Ca2+ or colchicine. The 35 K protein adsorbed on tubulin-Sepharose 4B was eluted by the addition of 2 mM ATP. ATP was shown to inhibit the interaction of 35 K protein with tubulin dimer or polymer. The 35 K protein was finally identified as glyceraldehyde 3-phosphate dehydrogenase from properties such as mobility on SDS-polyacrylamide gel electrophoresis, cleavage pattern on limited proteolysis, ability to bind to tubulin, and so on.
TL;DR: The maximum rate of Ca2+ release in the presence of ATP was considered to be sufficient to cause muscular contraction and is consistent with those reported in skinned muscle sarcoplasmic reticulum.
Abstract: Rapid Ca2+ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free Ca2+ concentration. Ca2+ efflux was activated by extravesicular Ca2+ with an apparent dissociation constant of 25 microM and was inhibited with an inhibition constant of 120 microM in the absence of Mg2+. Caffeine enhanced the Ca2+ release rate by increasing only the affinity of Ca2+ for the activation site. Mg2+ reduced the Ca2+ release rate by competitive binding to the activation site. ATP increased the Ca2+ release rate very much without changing the affinities of Ca2+ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca2+ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca2+ release in the presence of ATP reached 80 s-1. This value is considered to be sufficient to cause muscular contraction.
TL;DR: The results indicate that amino acid composition is well correlated to location in an organism, biological function, folding type, and disulfideonding, and the findings are discussed from the protein-taxonomical point of view.
Abstract: Correlations of the amino acid composition of a protein to its location in an organism, biological function, folding type, and disulfide bond(s) were examined for 356 proteins. In the present data set, 325 proteins of known location and biological characters were divided into 122 intracellular enzymes (BI), 73 intracellular non-enzymes (BII), 45 extracellular enzymes (BIII), and 85 extracellular nonenzymes (BIV). The composition of these proteins were expressed as points in the composition space of 18 orthogonal axes, each representing the content of an amino acid. The distributions of points of BI and BIII were narrow and approximately spherical but those of BII and BIV were distributed rather widely. The groups are separated from each other in the space. We divided the space into four regions (A1 to A4) corresponding to the groups BI to BIV. A protein could be assigned to one of the four groups (A1 to A4) from its amino acid composition: The proteins correctly assigned amounted to 177 out of 195 intracellular proteins, and 94 out of 130 extracellular proteins. The correspondence was about 80% for classification into intracellular and extracellular proteins and 66% for that into the four groups. The folding type also had a significant correlation to the above groups, i.e., intracellular enzymes are rich in alpha/beta, nonenzymes alpha, extracellular enzymes beta and alpha + beta, and nonenzymes beta. The differences in average composition between intra- and extracellular proteins, and between enzymes and nonenzymes were related to the structural characters, i.e., intracellular proteins contain more amino acids favoring alpha-helix than extracellular ones, and enzymes contain more hydrophobic amino acids than nonenzymes. The statistics on 213 Cys-containing proteins showed that disulfide bond(s) are found mostly (90%) in the extracellular proteins. The results indicate that amino acid composition is well correlated to location in an organism, biological function, folding type, and disulfide bonding. The implications of the new findings are discussed from the protein-taxonomical point of view, and the validity of the present method is assessed.
TL;DR: Bovine, human, and goat alpha-lactalbumins prepared by the ordinary methods were found to contain 1.1-1.3 atoms of Ca per protein molecule and removal of Ca2+ was shown to destabilize the tertiary structures in the three proteins.
Abstract: Bovine, human, and goat alpha-lactalbumins prepared by the ordinary methods were found to contain 1.1-1.3 atoms of Ca per protein molecule. Removal of Ca2+ was shown to destabilize the tertiary structures in the three proteins. The three apoproteins were indicated to change in the conformation by heat from the native-like to the unfolded state. Degree of restoration of the native tertiary structure in 5 mM Tris-HCl and 0.1 mM EDTA at pH 7.2 and 25 degrees C by addition of Ca2+ was determined from change in CD ellipticity at 270 nm, and the apparent binding constant of Ca2+ was analyzed to be 2.5 X 10(8) (bovine), 3.0 X 10(8) (human), and 2.8 X 10(8) M-1 (goat). Also, value of the binding constant of Ca2+ to the native-like apoform was estimated from the apparent binding constant and equilibrium constant of the conformational change of the apoform. The binding properties of Mn2+, Mg2+, and Zn2+ to the bovine protein at neutral pH are also discussed.
TL;DR: The binding of the chymotryptic Troponin T subfragments to tropomyosin, troponin I, and tropon in C was semiquantitatively examined and the binding sites of tropomyo- and tropo-binding sites were elucidated.
Abstract: The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.
TL;DR: The rate of Ca2+ release was markedly increased by AMP; the dependence of the rate on AMP concentration followed a Michaelis-Menten type equation with a Hill coefficient of 1 and an apparent affinity for AMP of about 2 mM.
Abstract: Fragmented sarcoplasmic reticulum (FSR) from rabbit skeletal muscle was passively loaded with 45Ca2+. Its Ca2+-induced Ca2+ release was measured in the presence of 0.1 M KCl and 5 mM MgCl2 at 0 degrees C by Millipore filtration. The following results were obtained. 1. The amounts of Ca2+-induced Ca2+ release from heavy SR, light SR, and unfractionated SR were 80, 20, and 60% of the amounts of preloaded Ca2+, respectively. Therefore, the experiments were carried out with unfractionated FSR. 2. The Ca2+-induced Ca2+ release from FSR was inhibited by procaine, but unaffected by caffeine and trifluoperazine. The rate of Ca2+ release decreased markedly with decreasing pH. 3. Various adenine nucleotides (ATP, AMPPNP, ADP, AMP) accelerated the Ca2+ release, and the accelerating effect was reversible. CTP had no effect on the release, but inhibited the accelerating effect of AMPPNP. 4. In the presence of 15 microM external free Ca2+, the final amount of the Ca2+ release was unaffected. The rate of Ca2+ release was markedly increased by AMP; the dependence of the rate on AMP concentration followed a Michaelis-Menten type equation with a Hill coefficient of 1 and an apparent affinity for AMP of about 2 mM. 5. In the presence of AMP, the amount of Ca2+ released increased, while the relative rate was unaffected by increasing the external Ca2+ concentration. The final amount released increased from 0 to 60% of the amount of preloaded Ca2+ by increasing the free Ca2+ concentration from 0.06 to 0.24 microM. The effect of external Ca2+ on the release was reversible. 6. The ratio between the amount of preloaded Ca2+ and that of Ca2+ release was independent of the Ca2+ concentration used for preloading. Furthermore, the dependence of the final amount of Ca2+-induced Ca2+ release on external Ca2+ was unaffected by internal Ca2+.
TL;DR: It is found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and C d-BP2 contains no labile sulfides.
Abstract: Two kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (l). It was also reported that Cd-BP1 and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2). Now, we have found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and Cd-BP2 contains no labile sulfide. The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BP1. Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BP1 is the first instance in this field.
TL;DR: The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200 and showed 50% homology with that of STI, but not bovine alpha-chymotrypsin.
Abstract: The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.
TL;DR: Human pancreatic phospholipase A2 was purified to homogeneity from pancreatic juice and a reliable radioimmunoassay for the enzyme was developed and was highly sensitive, reproducible and specific.
Abstract: Human pancreatic phospholipase A2 was purified to homogeneity from pancreatic juice and a reliable radioimmunoassay for the enzyme was developed. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 14,000. Phosphatidylcholine was hydrolyzed well in an alkaline pH range, and the optimum activity was obtained at pH 9. Calcium ion was indispensable for activity. The enzyme was stable to heat treatment at 60 degrees C for 5 min. The radioimmunoassay system was highly sensitive, reproducible and specific. The dilution curves for the sera of patients with acute pancreatitis were parallel to the standard curve. In healthy individuals, serum phospholipase A2 concentrations ranged from 2.0 to 7.9 ng/ml, the average being 5.1 ng/ml (S.D.: 1.7). In patients with acute pancreatitis, significant elevations of serum phospholipase A2 contents were observed, and the highest value found was 4,000 ng/ml.
TL;DR: The results indicated are very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.
Abstract: A post-proline cleaving enzyme [prolyl endopeptidase, EC 3.4.21.26] was purified about 3,700-fold from an extract of bovine brain by a series of column chromatographies on DEAE-Sephadex, hydroxyapatite and PCMB-T-Sepharose, and gel filtration on Sephadex G-200 using N-carbobenzoxy-Gly-Pro-beta-naphthylamide (Z-Gly-Pro-2-NNap), thyrotropin releasing hormone (TRH) and oxytocin as substrates. The purified enzyme appeared homogeneous as judged by disc gel and SDS gel electrophoreses. The enzyme was most active at pH 7.5 and 7.2 with Z-Gly-Pro-2-NNap and TRH, respectively, and hydrolyzed peptide bonds involving Pro-X (X=amino acid, peptide, ester and amide) bonds of synthetic substrates, oxytocin, vasopressin, neurotensin, substance P, tuftsin, bradykinin, and insulin B chain. However, the enzyme was inert toward collagen, gelatin, and casein. The enzyme was completely inactivated by diisopropylphosphorofluoridate (DFP), Z-Gly-Pro-chloromethyl ketone and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenylmethane sulfonylfluoride (PMSF) or metal chelators. Determination of the amino acid composition revealed that the enzyme contained 25 half-cystines. Modification of three cysteine residues of the enzyme by PCMB led to complete inactivation. The isoelectric point of the enzyme was 4.8, and the molecular weight was estimated to be 76,000 by ultracentrifugal analysis and 75,000-74,000 by both gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is present as a monomer. These results indicate that the post-proline cleaving enzyme from bovine brain is very similar to those previously purified from lamb brain and kidney in its enzymatic properties, substrate specificity and physicochemical properties, in sharp contrast with the results obtained by Tate, who reported that the bovine brain prolyl endopeptidase was inert toward oxytocin, vasopressin and bradykinin.
TL;DR: The selected ion monitoring (SIM) technique was applied to determination of platelet-activating factor (PAF) or acetyl glyceryl ether phosphorylcholine (AGEPC) and two types of PAF were found in human neutrophils on the challenge with ionophore A23187.
Abstract: The selected ion monitoring (SIM) technique was applied to determination of platelet-activating factor (PAF) or acetyl glyceryl ether phosphorylcholine (AGEPC). Two types of PAF, 1-hexadecyl- and 1-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (C16 = 0 AGEPC and C18 = 0 AGEPC), were found in human neutrophils on the challenge with ionophore A23187. The contents of C16 = 0 AGEPC in 1 X 10(7) neutrophil cells of four volunteers, respectively, were 47, 18, 59, and 73 ng and those of C18 = 0 AGEPC were 22, 4, 19, and 31 ng.
TL;DR: One form of CANP highly sensitive to calcium ions was purified by column chromatographic procedures to homogeneity as mentioned in this paper, and the purified enzyme required microM order Ca2+ (mu CANP), and the half maximum activity was attained at 50 microM Ca2+.
Abstract: One form of calcium-activated neutral protease (CANP) highly sensitive to calcium ions was purified by column chromatographic procedures to homogeneity. The purified enzyme required microM order Ca2+ (mu CANP), and the half-maximum activity was attained at 50 microM Ca2+. The electrophoretic mobility in a non-denaturing buffer showed that this enzyme is less acidic than another CANP which required mM order Ca2+ (mCANP). On SDS-polyacrylamide gel electrophoresis, the enzyme separated into two components with molecular weights of 79,000 and 28,000, respectively. Of these, the former was slightly larger than the counterpart of mCANP (Mr 76,000). Thus, mu CANP cannot be derived from mCANP by limited autolysis.
TL;DR: Analysis of the groups in terms of various characters of proteins, such as source, molecular size, location in an organism, biological function, etc, revealed that the groups have strong correlations to the location (inside or outside the cell) and functional character (enzyme or nonenzyme).
Abstract: Data on amino acid composition were collected in order to classify proteins into groups. The composition of a protein is expressed as a point in an orthogonal coordinate system, taking fractions of amino acids along 18 axes, which represent 18 amino acids (we use Asx and Glx for the sum of Asp and Asn and that of Glu and Gln, respectively). Thus, proteins of known amino acid compositions (356 single polypeptides chains) are distributed as points in this composition space. Since the radial distribution of the points from the origin (the average composition) did not show any distinct separation into groups, we checked the angular distribution of points in the space. Thirteen groups were found by a computer method based on the density. Analysis of the groups in terms of various characters of proteins, such as source (eukaryote or prokaryote), location in an organism, biological function, etc. revealed that the groups have strong correlations to the location (inside or outside the cell) and functional character (enzyme or nonenzyme). Also, the presence of disulfide bond(s) seems to be characteristic of extracellular proteins. Protein source, molecular size and ability to form an oligomer had little correlation to the grouping. Therefore, proteins may be classified into four types as follows: BI, intracellular enzymes; BII, intracellular nonenzymes; BIII, extracellular enzymes; and BIV, extracellular nonenzymes.
TL;DR: An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells and was assessed using the sensitivity to micrococcal nuclease.
Abstract: An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells. The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation. In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons. When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented. The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts. Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction. The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease.
TL;DR: It was established that the binding site for Q beta replicase is determined by nucleotide sequences that are located near the middle of MDV-1 RNA, and it was indicated that the 3'-terminal region of the template is required for the initiation of RNA synthesis.
Abstract: Fragments of MDV-1 RNA (a small, naturally occurring template for Q beta replicase) that were missing nucleotides at either their 5' end or their 3' end were still able to form a complex with Q beta replicase. By assaying the binding ability of fragments of different length, it was established that the binding site for Q beta replicase is determined by nucleotide sequences that are located near the middle of MDV-1 RNA. Fragments missing nucleotides at their 5' end were able to serve as templates for the synthesis of complementary strands, but fragments missing nucleotides at their 3' end were inactive, indicating that the 3'-terminal region of the template is required for the initiation of RNA synthesis. The nucleotide sequences of both the 3' terminus and the central binding region of MDV-1 (+) RNA are almost identical to sequences at the 3' terminus and at an internal region of Q beta (-) RNA.
TL;DR: A phylogenetic tree of legume double-headed inhibitors was constructed on the basis of the matrix of amino acid differences among their sequences because B-II possessed an extremely different amino acid sequence from those of the other peanut inhibitors and was thought to be biosynthesized from a gene different from that of theother inhibitors.
Abstract: The amino acid sequences of four peanut protease inhibitors (A-I, A-II, B-I, and B-II) were determined by conventional methods and by comparison of peptide maps of their tryptic digests with that of B-III on HPLC. A-I, A-II, B-I, and B-III had the same amino acid sequence except for differences in their N-terminal regions. This suggests that the four inhibitors would be derived from an original inhibitor with a longer N-terminal amino acid sequence by proteolysis of its N-terminal region. But B-II possessed an extremely different amino acid sequence from those of the other peanut inhibitors and was thought to be biosynthesized from a gene different from that of the other inhibitors. A phylogenetic tree of legume double-headed inhibitors was constructed on the basis of the matrix of amino acid differences among their sequences. The double-headed inhibitors whose sequences have been determined were classified into four groups.
TL;DR: The purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography.
Abstract: Purification of cytoplasmic dynein from unfertilized sea urchin eggs was performed in the presence of protease inhibitors to avoid proteolysis throughout the purification procedure, which comprised several chromatographies, including a calmodulin-Sepharose 4B affinity column chromatography. This is the first report of the purification of cytoplasmic dynein to near homogeneity. The purified fraction was composed of a single high molecular weight polypeptide and some minor low molecular weight polypeptides. The high molecular weight polypeptide comigrated with flagellar dynein A beta chain from sperm on SDS-polyacrylamide gels. There was no polypeptide stainable with periodic acid-Schiff reagent (PAS) in the purified cytoplasmic dynein fraction. Cytoplasmic dynein showed characteristics quite similar to those of axonemal dynein, i.e. high substrate specificity for ATP and inhibition by low concentrations of vanadate, though its Ca-ATPase activity showed almost the same dependence on the concentration of either divalent cations or KC1 as the Mg-ATPase activity. The purified enzyme seemed to possess functional form as judged from its properties: 1) pH dependence of the ATPase activity, 2) dependence of the ATPase activity on MgCl2 and KCl concentration, 3) Km for Mg-ATP, and 4) binding to flagellar doublet microtubules. Cytoplasmic dynein bound to calmodulin-Sepharose 4B only in the presence of Ca2+, and was eluted with EGTA. Furthermore, the ATPase activity was enhanced 6-fold by calmodulin in a Ca2+-dependent manner. The activation by calmodulin was prevented by a stoichiometric amount of trifluoperazine.
TL;DR: It was found that fodrin purified from porcine brains co-sedimented with microtubules which were assembled from phosphocellulose-purified tubulin prepared from porCine brains, which indicates thatfodrin bound to micro Tubule-associated proteins (MAPs), and raises the possibility that micro Tubules are involved in the submembranous cytoskeleton of neurons with Fodrin.
Abstract: Fodrin is a spectrin-like protein present in the cortical cytoplasm of neurons and binds to F-actin to induce gelation of actin. We found that fodrin purified from porcine brains co-sedimented with microtubules which were assembled from phosphocellulose-purified tubulin prepared from porcine brains. This indicates that fodrin bound to microtubules. An unusual enhancement of turbidity at 350 nm was observed when microtubules were assembled in the presence of fodrin. Microscopic observations showed that fodrin bundled the microtubules. The interaction between fodrin and microtubules was decreased by microtubule-associated proteins (MAPs), indicating that fodrin and MAPs interacted with microtubules competitively. These data raise the possibility that microtubules are involved in the submembranous cytoskeleton of neurons with fodrin.
TL;DR: Enzymic characterization showed that the lysosomal membrane preparation isolated by this procedure was almost free from mitochondria, peroxisomes, and endoplasmic reticulum, and the electrophoretic profiles of both membrane preparations were quite similar.
Abstract: A rapid and simplified method for the preparation of lysosomal membranes from rat livers was developed. Enzymic characterization showed that the lysosomal membrane preparation isolated by this procedure was almost free from mitochondria, peroxisomes, and endoplasmic reticulum. Acid phosphatase was used as a marker enzyme for lysosomal membrane, because about 40% of the total acid phosphatase activity in the lysosomes was associated with the membranes. With this method, the yield of the purified membrane was 115 micrograms/g wet weight of liver and the relative specific activity of acid phosphatase in the purified membrane was 105-110. On SDS-polyacrylamide gel electrophoretic analysis, the purified membrane revealed glycoprotein bands in the region of M.W. between 60,000 and 110,000, which were characteristic of the tritosomal membrane. Our lysosomal membrane preparation was compared with lysosomal membranes prepared from normal rat liver lysosomes isolated by the method of Wattiaux et al. [(1978) J. Cell Biol. 78, 349-368]. Although the specific activity of acid phosphatase in our membrane preparation was higher than that in the membrane prepared by the existing method, the electrophoretic profiles of both membrane preparations were quite similar.