A novel metalloproteinase originally isolated from brain myelin membranes is present in many tissues.

A monoclonal antibody, CG4, was raised to a novel 60 kDa metalloproteinase purified from a bovine brain myelin glycoprotein fraction. Glycoproteins extracted from both myelin and nine different bovine tissues showed the 60 kDa CG4-immunoreactive band by immunoblotting in amounts that broadly paralleled enzymic activity of this metalloproteinase and varied relatively little among the tissues.     

Isolation and characterization of two novel peptide amides originating from myelin basic protein in bovine brain

During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-amide and Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-amide. These 12 and 16 residues peptides had the primary structure identical to the N-terminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and-16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain.     

Isolation and partial characterization of chick brain synaptic plasma membranes.

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones, D. H. and Matus, A. I. (1974) Biochim. Biophys. Acta 356, 276-287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organelles. Fraction 2 was recovered from the 28.5-34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na+ +K+)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5'-nucleotidase (EC 3.1.3.5) were, respectively, 4.5, 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation and characterization of a novel fucoganglioside of human erythrocyte membranes.

A slow migrating monosialosyl ganglioside with blood group H activity was isolated from human erythrocyte membranes and identified as monosialosylceramide decasaccharide. Its structure was determined by degradation with exo- and endoglycosidases, by mass spectrometric characterization of a nonasaccharide liberated by endo-beta-galactosidase, and by methylation analysis and mass spectrometry of permethylated glycolipids before and after enzymatic degradation. The ganglioside contains two oligosaccharide chains branched through GlcNAcbeta1 leads to 6(GlcNAcbeta1 leads to 3) Gal structure, and the terminal Gal of the 1 leads to 6-linked oligosaccharide was fucosylated, whereas that of the 1 leads to 3-linked oligosaccharide was sialosylated as seen below.     

A novel proteinase associated with mitochondrial membranes

Mitochondrial inner membranes catalyzed the endoproteolytic release of radioactively labelled polypeptide fragments of ¹²⁵I labelled insulin covalently attached to agarose beads. Demonstration of this proteolytic activity was dependent upon detergent and inhibited by a variety of proteinase and sulfhydryl group inhibitors. Unlike three previously described peripheral mitochondrial proteinases, the insulin hydrolyzing mitochondrial proteinase was resistent to extraction with high salt but solubilized with a non-ionic detergent, indicating its integral association with the membrane.     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Isolation and partial characterization of chick brain synaptic plasma membranes

An isolation procedure for synaptic plasma membranes from whole chick brain is reported that uses the combined flotation-sedimentation density gradient centrifugation procedure described by Jones and Matus (Jones. D. H. and Matus. A. I. (1974) Biochim. Biophys. Acta 356, 276–287) for rat brain. The particulate of the osmotically shocked and sonicated crude mitochondrial fraction was used for a flotation-sedimentation gradient step. Four fractions were recovered from the gradient after 30 min centrifugation. The fractions were identified and characterized by electron microscopy and by several markers for plasma membrane and other subcellular organcelles. Fraction 2 was recovered from the 28.5–34% (w/v) sucrose interphase and contained the major part of the activities of the neuronal plasma membrane marker enzymes. The specific activities of the (Na⁺+K⁺)-activated ATPase (EC 3.6.1.3), acetylcholinesterase (EC 3.1.1.7) and 5′-nucleotidase (EC 3.1.3.5) were, respectively, 4.5. 2.0 and 1.2 times higher than in the homogenate. However, Fraction 2 also contained considerable amounts of activities of putative lysosomal and microsomal markers in addition to lower amounts of mitochondrial and myelin markers. Although no prepurification of synaptosomes from the crude mitochondrial fraction was performed, the synaptic plasma membranes obtained showed many properties analogous to similar preparations from rat brain described in recent years.     

Isolation and characterization of a novel peptide amide from porcine brain.

Peptide with C-terminal tyrosine amide was isolated from porcine brain by acid extraction and sequential steps of reverse phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structure: Ac-Ala-Ser-Glu-Lys-Arg-Pro-Ser-Glu-Arg-His-Gly-Ser-Lys- Tyr-amide. Since this peptide had the identical sequence to N-terminus of porcine myelin basic protein (pMBP) 1-14, we have designated porcine myelin peptide amide 14 (pMPA14). The final HPLC step yielded 20 micrograms of homogeneous peptide preparation from 20 kg brain tissue. Unlike other amidated peptides, pMPA14 may be produced by non enzymatic mechanism or unknown amidating enzyme. This unique amidation seems to occur exclusively to MBP in the brain.     

Mammalian brain plasma membranes : Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na⁺-K⁺) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.     

Isolation and partial characterization of a protein with HMG-CoA reductase phosphatase activity associated with rat liver microsomal membranes.

Several rat liver HMG-CoA-reductase (HMG-CoA-Rd) phosphatase activities have been shown to be associated with the endoplasmic reticulum. These activities were not due to glycogen contamination, as judged not only from different patterns of solubilization of the microsomal membranes and the glycogen pellet but also by differential centrifugation behavior under standard conditions and in a sucrose gradient. We present evidence that at least three forms of protein phosphatase are associated with microsomal membranes: a polycation-stimulated type 2A phosphatase, a type 2C phosphatase, and a non-2A, non-2B, non-2C phosphatase. This last HMG-CoA-Rd phosphatase activity corresponding to an 85 kDa protein was partially purified by several chromatographic procedures. The IC50 value for the inhibition of the HMG-CoA-Rd phosphatase by I-2 was 10-fold higher than for the inhibition of the purified type 1 catalytic subunit from rabbit skeletal muscle. The microsomal HMG-CoA-Rd phosphatase activity was slightly affected by the protein inhibitor that inhibits type 2A activity when HMG-CoA reductase is the substrate. The HMG-CoA-Rd phosphatase activity is spontaneously active and it is not reactivated in the presence of Mg2+ or polycations. The holoenzyme does not contain the inhibitor-2 and it is not reactivated by incubation with ATP and glycogen synthase kinase-3. Proteolytic treatment of the enzyme yielded a polypeptide fragment of low Mr (37 kDa) with reduced activity. A model of holoenzymatic HMG-CoA-Rd phosphatase and its relation to the microsomal membranes is presented.     

Identification and characterization of SppA, a novel light-inducible chloroplast protease complex associated with thylakoid membranes.

A new component of the chloroplast proteolytic machinery from Arabidopsis thaliana was identified as a SppA-type protease. The sequence of the mature protein, deduced from a full-length cDNA, displays 22% identity to the serine-type protease IV (SppA) from Escherichia coli and 27% identity to Synechocystis SppA1 (sll1703) but lacks the putative transmembrane spanning segments predicted from the E. coli sequence. The N-terminal sequence exhibits typical features of a cleavable chloroplast stroma-targeting sequence. The chloroplast localization of SppA was confirmed by in organello import experiments using an in vitro expression system and by immunodetection with antigen-specific antisera. Subfractionation of intact chloroplasts demonstrated that SppA is associated exclusively with thylakoid membranes, predominantly stroma lamellae, and is a part of some high molecular mass complex of about 270 kDa that exhibits proteolytic activity. Treatments with chaotropic salts and proteases showed that SppA is largely exposed to the stroma but that it behaves as an intrinsic membrane protein that may have an unusual monotopic arrangement in the thylakoids. We demonstrate that SppA is a light-inducible protease and discuss its possible involvement in the light-dependent degradation of antenna and photosystem II complexes that both involve serine-type proteases.     

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.     

Isolation and characterization of Neurospora crassa plasma membranes.

The isolation and characterization of plasma membranes from a cell wall-less mutant of Neurospora crassa are described. The plasma membranes are stabilized against fragmentation and vesiculation by treatment of intact cells with concanavalin A just prior to lysis. After lysis, the concanavalin A-stabilized plasma membrane ghosts are isolated by low speed centrifugation techniques and the purified ghosts subsequently converted to vesicles by removal of the bulk of the concanavalin A. The yield of ghosts is about 50% whereas the yield of vesicles is about 20%. The isolated plasma membrane vesicles have a characteristically high sterol to phospholipid ratio, Mg2+-dependent ATPase activity and (Na+ plus K+)-stimulated Mg2+ATPase activity. Only traces of succinate dehydrogenase and 5'-nucleotidase are present in the plasma membrane preparations.     

A novel fluorescent probe that is brain permeable and selectively binds to myelin.

Myelin is a multilayered glial cell membrane that forms segmented sheaths around large-caliber axons of both the central nervous system (CNS) and peripheral nervous system (PNS). Myelin covering insures rapid and efficient transmission of nerve impulses. Direct visual assessment of local changes of myelin content in vivo could greatly facilitate diagnosis and therapeutic treatments of myelin-related diseases. Current histologic probes for the visualization of myelin are based on antibodies or charged histochemical reagents that do not enter the brain. We have developed a series of chemical compounds including (E,E)-1,4-bis(4'-aminostyryl)-2-dimethoxy-benzene termed BDB and the subject of this report, which readily penetrates the blood-brain barrier and selectively binds to the myelin sheath in brain. BDB selectively stains intact myelinated regions in wild-type mouse brain, which allows for delineation of cuprizone-induced demyelinating lesions in mouse brain. BDB can be injected IV into the brain and selectively detect demyelinating lesions in cuprizone-treated mice in situ. These studies justified further investigation of BDB as a potential myelin-imaging probe to monitor myelin pathology in vivo.     

Isolation and characterization of cell membranes

The steps outlined above should provide a framework for developing and evaluating a scheme for isolating a particular membrane system of interest. Further characterization often focuses on the specific research interests of an investigator. Most subsequent techniques will probably involve biochemical analyses of protein and lipid components and biophysical analyses of membrane properties. The techniques to be used, such as SDS gel electrophoresis, enzyme kinetics, receptor binding, protein purification, etc. are not unique to membrane systems and will not be described in detail. However, it is likely that the functional relevance and success of subsequent analyses will be aided if the starting membranes are as homogeneous and well-characterized as possible. Even if some of the fractionation steps are bypassed, such as might occur if a specific membrane protein is detergent-extracted, purified, and reconstituted directly from a crude homogenate, a full understanding of membrane protein function will probably require complementary studies on intact cells and isolated membranes.     

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.     

Purification and characterization of a novel plant metalloproteinase

A novel enzyme, the first metalloproteinase purified from a monocotyledonous plant, was extracted from the endosperm of sorghum seedlings and purified to homogeneity by ion exchange chromatography and size exclusion chromatography. SDS-PAGE analysis reveals a dimeric 17-kDa protein with two 8-kDa subunits linked by disulfide bond(s). The enzyme is 97% inhibited by 1 mM EDTA and is unaffected by inhibitors of aspartic, cysteine, and serine proteinases. Its pH optimum is 7.0 with hemoglobin as substrate.     

Isolation and characterization of plasma membranes from Friend erythroleukaemic cells. A study with sphingomyelinase C

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (I) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6-7% of phospholipid, 8-9% of cholesterol and 12-15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15-20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C (S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.     

ADP-ribosyltransferase activity in myelin membranes isolated from human brain

An ADP-ribosyltransferase has been identified in compact myelin and in several white matter fractions which contain less compact myelin, fractionated on the basis of increasing protein/lipid ratios. One fraction the P₃A contained the greatest activity although the activity in compact myelin was only slightly less. The ADP-ribosyltransferase activity of solubilized myelin was stimulated by increasing amounts of GTPS and was specific for the -isomer of NAD. Although ADP-ribosylation was demonstrated with the heterotrimeric G proteins in the 40–50 kDa range, the substrate for the ADP-ribosyltransferase in the 20 kDa range was identified as MBP. ADP-ribosyltransferase; myelin basic protein; signal transduction.Abbreviations ADP-ribose adenosine diphosphate ribose - APAD 3-acetylpyridine adenine dinucleotide - ATP adenosine triphosphate - C-1, 2, 3 etc MBP components isolated by CM52 chromatography - EDTA ethylenediaminetetraacetic acid - GTP guanosine triphosphate - GTPS guanosine 5-(3-0-thio)triphosphate - INH isonicotinic acid hydrazide - MBP myelin basic protein - NAD nicotinamide adenine dinucleotide - PMSF phenylmethylsulfonyl fluoride - PLP proteolipid protein Special issue dedicated to Dr. Leon S. Wolfe.     

A new protein of the brain myelin: isolation and chemical characterization

An unknown protein has been isolated from bovine brain myelin. This protein, purified in the nonionic detergent n-octylpolydisperse oligooxyethylene, reveals on SDS gel electrophoresis a large number of bands in the higher MW region. However, chemical analysis and gel chromatography indicate the presence of a single, small protein containing large amounts of bound phosphatidylserine. N-terminal and C-terminal sequences, aminoacid composition, and the anomalous electrophoretic behaviour led us to exclude the protein as a fragment of other already known myelin proteins.