The effect of phenobarbital and amidopyrine on the rate of decomposition of isoforms of cytochrome P-450 in mouse liver microsomes

Separation of microsomal proteins in gradient polyacrylamide gel gives 60 protein bands. The molecular mass range of 48-58 kDa corresponding to cytochrome isozymes contains 7 bands for intact mice and 8 bands for phenobarbital-induced mice. Phenobarbital treatment causes both the appearance of a new cytochrome P-450 isozyme with a molecular mass of 56 kDa and the increase in the content of three isozymes with molecular masses of 54, 52.5 and 50 kDa. The half-life time of cytochrome P-450 isozymes in the livers of intact and phenobarbital-induced mice differs from 15 to 42 hours. Phenobarbital induction results in the breakdown acceleration of the isozyme with a molecular mass of 52.5 kDa and the breakdown retardation of the isozyme with a molecular mass of 54 kDa. Aminopyrine injections to phenobarbital-pretreated mice result in the breakdown acceleration of the cytochrome P-450 isozyme with a molecular mass of 56 kDa.     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.     

Isolation and partial characterisation of immunoglobulin from southern bluefin tuna Thunnus maccoyii Castelnau

Specific and total serum immunoglobulins were extracted by immunoaffinity, mannan-binding protein and Protein A affinity chromatography from southern bluefin tuna (Thunnus maccoyii Castelnau) immunised with rabbit IgG, and from non-immunised southern bluefin tuna. SDS-PAGE in 10% reducing gels revealed two heavy chains with molecular weights of approximately 74.6 +/- 1.3 kDa and 71.2 +/- 0.9 kDa, and two light chains with molecular weights of approximately 29 +/- 1.2 kDa and 28 +/- 1.0 kDa. Under non-reducing, but denaturing, conditions in 4% and 5% SDS-PAGE gels, a high molecular weight and a low molecular weight fraction were demonstrated. By gel filtration using Sephacryl HR 300 a molecular weight of 845 kDa, consistent with a tetramer, was obtained for the high molecular weight fraction, and a molecular weight of 168 kDa, consistent with a monomer, was obtained for the low molecular weight fraction. The extinction coefficient at A280 for the purified immunoglobulin (Ig) was determined to be 1.24. Tuna a-rabbit IgG Ig was reactive with all non-reduced mammalian IgG antigens tested, suggesting that common conformational antigenic determinants were recognised.     

Identification of pro-epidermal growth factor and high molecular weight epidermal growth factors in adult mouse urine

By use of immunoblot analysis, we demonstrate the presence of a pro-Epidermal growth factor (EGF) with an approximate molecular weight of 165 kDa in adult mouse urine. In addition, urine contains four high molecular weight EGFs with approximate molecular weights of 116, 97, 66 and 56 kDa. The 165 kDa pro-EGF as well as the 66 and 56 kDa EGFs also are detectable in mouse kidney extract. Neither urine nor kidney contain the mature EGF of 6 kDa. The 165 kDa pro-EGF is the major product synthesized in renal tissue and secreted in urine. The finding of high molecular weight EGFs in urine suggests that part of pro-EGF secreted into urine undergoes partial proteolysis distal to its site of synthesis.     

Neurotoxin 6-aminonicotinamide affects levels of soluble proteins and enzyme activities in various tissues of golden hamsters

The effects of neurotoxin 6-aminonicotinamide (6-AN) on the levels of soluble proteins and enzyme activities in various tissues of golden hamsters were investigated. SDS-polyacrylamide gel electrophoresis showed that a soluble spinal cord protein with molecular mass 75.0 kDa was present at a higher concentration in the treated group compared to that in the control while that of a molecular mass 64.8 kDa appeared to be missing. However, there were no noticeable differences in protein concentrations observed with the cerebrum, brain stem, and cerebellum. Similarly, treatment with 6-AN decreased the concentration of a soluble protein in pectoral muscle having molecular mass 97.2 kDa and increased those having molecular masses 207.4 and 32.1 kDa. In the kidney, soluble proteins with molecular masses 176.6 kDa was missing and those of molecular masses 97.6, 49, 43.3, and 33.8 kDa were decreased whereas those of molecular masses 64.7 and 33.1 kDa were increased. In the testis the soluble proteins with molecular masses 125.4, 88.7, 69.0, 31.2, 19.1, and 17.4 kDa were missing and those of molecular masses 97.0, 51.3, 42.0, 33.0, 27.2, and 22.6 kDa were present in lower amounts whereas those of molecular masses 311.5, 75.0, 64.0, 54.1, and 53.2 kDa were present in higher amounts. The specific activity of 6-phosphogluconate dehydrogenase was markedly increased in the liver but that of other tissues was not affected. Acetylcholinesterase activity was markedly reduced in the spleen but was enhanced in the intestine. Monoamine oxidase activity was markedly reduced in the brain stem, cerebrum, kidney, and liver. The results suggest that the changes in levels of soluble proteins and enzyme activities shown with golden hamster tissues by 6-AN administration were quite different from those shown with quail tissues.     

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

Wall mannoproteins of the yeast and mycelial cells of Candida albicans: nature of the glycosidic bonds and polydispersity of their mannan moieties

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7% protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2.5 kDa N-glycosidically linked sugar chain and a 31.5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34, 30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.     

Immunochemical and immunohistochemical studies, using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13-17 kDa nonamelogenins, on immature enamel of the pig and rat

Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13-17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149-173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1-148 of the antigen. Immunoelectro-transfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13-17 kDa nonamelogenins. The antiserum against the 13-17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13-17 kDa nonamelogenins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate-binding proteins of tumor lines with different growth properties

Summary A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weights of 80 kDa, 70 kDa, 22 kDa, 18 kDa and 16 kDa and of a fucose-binding protein at apparent molecular weight of 42 kDa. The -glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5–8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5–8#1 and 5–20#20, whereas the metastatic variant 5–20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.     

Mechanism of hypoglycaemic action of methylenecyclopropylglycine.

Cytochrome b-245 from neutrophil plasma membranes contains two types of subunit with apparent molecular masses from gel electrophoresis in the presence of SDS of 23 kDa and 76-92 kDa. Radiation-inactivation analysis revealed a single-exponential decay process for the visible absorption of the haem chromophore in the membrane, corresponding to a molecular mass of 21 +/- 5 kDa for the haem-containing polypeptide chain. Sedimentation equilibrium of the cytochrome solubilized by the detergent Triton N101 showed that the protein was polydisperse, with a molecular mass of approx. 350 kDa for the smallest detectable species. In another detergent, n-octyl beta-O-glucopyranoside (octyl glucoside), the molecular mass of the haem-containing particle was found to be 20-30 kDa. Thus the quaternary structure of the protein breaks down in this detergent. The haem group is inferred to be attached to the smaller subunit.     

Dextran retention in the rat brain following release from a polymer implant.

Intracranial controlled release polymers may improve drug administration to the brain, where therapy is frequently limited due to the low permeability of brain capillaries to therapeutic agents. On the basis of drug transport and elimination rates, we proposed that high molecular weight, water-soluble molecules would be retained in the brain space following release from an intracranial implant. To test this hypothesis, solid particles of different molecular weight fractions of fluorescein isothiocyanate labeled dextran (FITC-dextran; 4 x 10(3) Da (4 kDa) < weight-averaged molecular weight (Mw) < 150 kDa) or fluorescein were uniformly dispersed in matrices of a polyanhydride copolymer synthesized from a fatty acid dimer and sebacic acid in a 50:50 ratio, P(FAD:SA). When incubated in buffered saline, FITC-dextran fractions of 70 kDa Mw were released from the polymer within 48 h; 4 kDa Mw FITC-dextran and fluorescein were released more slowly. Following implantation of P(FAD:SA) matrices containing either 70 kDa Mw FITC-dextran, 4 kDa Mw FITC-dextran, or fluorescein into the brains of normal rats, fluorescent tracers were continuously released into the brain tissue for 30 days. Tracer concentrations within the brain were significantly higher for large molecular weight tracers (70 kDa Mw FITC-dextran > 4 kDa Mw FITC-dextran > fluorescein). The rate of elimination, kapp, of each tracer from the brain was determined by comparing experimental data with a model describing tracer diffusion/elimination in the brain extracellular space; kapp decreased with increasing molecular weight (fluorescein > 4 kDa Mw FITC-dextran > 70 kDa Mw FITC-dextran).     

Separation and purification of high molecular weight glycoproteins using agarose gel electrophoresis.

Several components of the extracellular matrix in the molecular weight range of 220 kDa to 150 kDa were purified by preparative electrophoresis on 2.5% Pro-Sieve agarose gels. These high molecular weight glycoproteins, separated under reducing conditions, were recovered in solution by extraction of individual agarose gel slices and analyzed on sodium dodecyl sulfate polyacrylamide gels and Western blots. This simple method permitted the separation and recovery of the laminin B chains (220 kDa and 205 kDa) and entactin (150 kDa) and may prove useful for the purification of other high molecular weight species.     

Subunit composition of the H+-ATPase complex from anaerobic bacterium Lactobacillus casei

The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.     

Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed     

西伯利亚鲟致病性嗜水气单胞菌外膜蛋白及其抗原性分析

采用十二烷基肌氨酸钠(Sarkosyl)法提取西伯利亚鲟嗜水气单胞菌(Aeromonas hydrophila)外膜蛋白,电泳显示所提取的主要外膜蛋白分子量为26～120 kDa;为比较该菌株与气单胞菌菌属其他细菌外膜蛋白组分及抗原性异同,以致病性豚鼠气单胞菌(A.caviae)、温和气单胞菌(A.sobria)和无致病力的嗜水气单胞菌为对照,电泳图谱显示4种气单胞菌外膜蛋白的分子量主要集中在26～120 kDa之间;利用抗西伯利亚鲟嗜水气单胞菌血清的免疫印迹试验表明该菌株外膜蛋白中分子量为75 kDa、52 kDa、43 kDa、40 kDa、34 kDa、28 kDa的蛋白条带呈现阳性反应,其他3种气单胞菌外膜蛋白中均有与该抗血清反应的条带,且分子量为28 kDa、34 kDa的反应条带为4株菌共有;43 kDa与75 kDa反应条带为部分菌株共有.为进一步筛选和研究致病性气单胞菌的共同保护抗原提供参考.     

Two high molecular mass proteases from sea urchin sperm.

Two-types of high molecular mass proteases have been purified from sea urchin sperm using DEAE-Sephacel, hydroxylapatite and Superdex 200 column chromatography. Both proteases showed similar hydrolyzing activities toward synthetic peptides, but they differed in the molecular mass and peptide composition. One was probably identical to a proteasome (multicatalytic proteinase), judging from its molecular mass (650 kDa) and polypeptide composition. The other one was composed of several polypeptides with molecular masses ranging from 24 kDa to 125 kDa and its molecular mass was estimated as 950 kDa by gel filtration. These two proteases, however, were closely related to each other. Immunological studies revealed that the 950-kDa protease comprised at least five subunits of the 650-kDa protease.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Identification of DNA ligase I related polypeptides in three different human cells

Partial purification of the DNA ligase from three human tissues (liver, thymus and lymphoblasts) revealed that each cell type contains several different polypeptides bearing a DNA ligase I activity. Their apparent molecular weights estimated after SDS PAGE, 130 kDa, 100 kDa and 80 kDa, are in agreement with previous reports. These polypeptides are related by proteolysis to a single higher molecular weight protein of 200 kDa which does not show DNA ligase activity but that could be a preprotein.