Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trimeresurus flavoviridis venom

A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.     

Characteristics of myelin basic protein from dog spinal cord]

Some characteristics of the myelin basic protein from dog spinal cord were studied. The basic protein has a molecular weight of 18,000; the isoelectric point is 9.5. The amino acid composition of the protein was determined. Electrophoresis in 15% polyacrylamide gel revealed the heterogeneity of the basic protein. The EAE-activity of the basic protein was tested on guinea pigs.     

Uridine Transport and Metabolism in the Central Nervous System

Myelin and myelin-containing (P3) fractions were prepared from human white matter by discontinuous sucrose gradient centrifugation. The myelin isolated from each of the fractions of different densities was morphologically and biochemically distinct. Light myelin fractions consisted of compact, multilamellar myelin, whereas the denser fractions consisted predominantly of loose myelin with fewer lamellae. The amounts of both basic protein and lipophilin (proteolipid protein) were reduced in the denser fractions. In contrast, the high-molecular-weight components were elevated in the dense fractions. The lipid composition was similar in all the fractions studied. Analysis of basic protein by gel electrophoresis at pH 10.6 revealed differences in basic protein microheterogeneity among the fractions. The light myelin fraction was enriched in the more positively charged basic protein components (components 1, 2, and 3), whereas these components were reduced in the denser fractions. Myelin in the dense fractions was enriched in the more modified forms of basic protein (components 6, 7, and 8). The pattern of microheterogeneity was different for basic protein isolated from myelins of a 2-year-old and an adult brain; the former showed fewer components and mainly the most cationic species. On the other hand, the pattern of microheterogeneity of basic protein isolated from the different density gradient fractions was similar for both ages.     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

Phospholipid vesicle aggregation induced by human myelin basic protein

Human myelin basic protein isolated from the brains of individuals who died with multiple sclerosis was more potent in inducing the aggregation of egg phosphatidylcholine vesicles than was the basic protein isolated from the brains of normal individuals. The portion of myelin basic protein which bound to egg phosphatidylcholine vesicles was separated from the free protein by sucrose density gradient centrifugation. Similar amounts of basic protein from normal or from multiple sclerosis brains are bound to the lipid and no consistent differences in the N^G, N^G dimethyl-arginine content of the protein fractions have been found.     

Stoichiometric relation of protein components in cerebral myelin from different species

Abstract— In cerebral myelin from man, ox, rabbit, guinea pig and chicken, the amounts of proteolipid protein, basic protein and the fraction of further protein components were found to be present in a fixed ratio of 5·0: 3·5: 2·0 by weight. The molecular weights of 25,000 and 35,000 as obtained for the basic protein and proteolipid protein might indicate that cerebral myelin contains one molecule of basic protein per molecule of proteolipid protein. This fixed ratio of protein components was found to be changed in myelin from the PNS and in cerebral myelin from rat and carp, with their exceptional basic proteins. Using the polyacrylamide-gel electrophoresis it was possible to demonstrate that a homogeneous structural protein (the Folch-Lees proteolipid protein) constitutes about 50 percent of the total amount of myelin proteins in all species studied. An attempt was made to correlate myelin protein and lipid patterns from various species.     

In Vivo Methylation of an Arginine in Chicken Myelin Basic Protein

Abstract: The amino acid sequence around the sole methylarginine residue in chicken myelin basic protein was determined and was found to be similar to that previously reported for mammalian myelin basic protein. The ratio N ^G, N '^G-dimethylarginine: N ^G-monomethylarginine:arginine was approximately 1.3:0.9:1.0. No N ^G, N ^G-dimethylarginine was detected in the protein. The in vivo incorporation of methyl groups from [methyl-³H]methionine into methylarginines in myelin was found to occur readily in 2-day-old chickens. Radioactively labelled N ^G, N '^G-dimeth-ylarginine and N ^G-monomethylarginine in myelin were derived solely from myelin basic protein. Radioactivity was also incorporated into N ^G, N ^G-dimeth-ylarginine, although this was not derived from myelin basic protein. As N ^G-monomethylarginine was easily separated from the dimethylarginines, and as it was derived from myelin basic protein, it may be a good marker for myelin basic protein turnover in vivo. A time course study of the incorporation showed that radioactivity was incorporated into N ^G-monomethylarginine up to 6 h after injection, and decayed slowly, with an apparent half-life of about 40 days.     

The identification of threonine-95 as the major site of glycosylation in normal human myelin basic protein.

The enzymic transfer of N-acetylgalactosamine to myelin basic protein and that to peptides derived from basic protein were compared. Basic protein treated with pepsin and trypsin before glycosylation resulted in decreases of 7% and 23% in the amount of N-acetylgalactosamine transferred to the peptides respectively. However, digestion of basic protein had little effect on the sites glycosylated. It was found that Thr-95 was the major site for glycosylation in both the intact human basic protein and in the tryptic peptides.     

Covalent linkage of phospholipid to myelin basic protein: identification of serine-54 as the site of attachment

The peptide portion of the lipopeptide isolated from bovine myelin basic protein contained glycine, lysine, and serine in a 2:1:1 molar ratio as determined by amino acid analysis. The N-terminus of the peptide was determined to be glycine. The tetrapeptide Gly53-Ser-Gly-Lys56 was the only segment of myelin basic protein that matched the above two characteristics. This tetrapeptide is highly conserved among the myelin basic proteins sequenced so far. After the selective degradation of the lipopeptide, phosphoserine was identified in the acid hydrolysate, thus indicating that Ser-54 of myelin basic protein in bovine brain is the site of attachment of polyphosphoinositide. Interestingly, serine-54 of myelin basic protein can be phosphorylated by the endogenous protein kinase myelin. However, myelin basic protein phosphorylated by the catalytic subunit of an exogenous soluble protein kinase failed to produce radioactively labeled lipopeptide. Hence the endogenous enzymes of myelin are thought to be involved in the formation of the covalent linkage between polyphosphoinositide and myelin basic protein. The conservation in sequence suggests a possible important structural role for the "phospholipidation" of myelin basic protein.     

Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent.

The localization of proteins in myelin was studied by the use of a non-penetrating reagent. Tritiated 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with 3H-labelled 4,4'-diisothiocyano-2,2'-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

The influence of pH on the degradation of bovine myelin basic protein by bovine brain cathepsin

The degradation of bovine myelin basic protein by bovine brain cathepsin D (ED 3.4.23.5) was studied over a pH range of 2.75 - 6.0. Throughout this pH range pepstatin, an inhibitor of cathepsin D, prevented the degradation. The degradation at a pH away from the optimum of pH 3.5 was predictably slower, but also resulted in more restricted cleavage. Above pH 4.5 bovine basic protein peptide 1 - 42 was not degraded further to peptide 1 - 36 as occurs at pH 3.5. Additionally, at pH 5.5 another fragment of basic protein, peptide 1 - 91, persisted indicating that under certain basic protein as well as basic protein peptide 43 - 169 may be cleaved in the molecular region of basic protein around the phenylalanyl-phenylalanine residues at position 88 - 89. The small amount of peptides 1 - 91 and 92 - 169 detected at pH 5.5 suggests that the bond between residues 91 and 92 in intact basic protein is a minor cleavage site. The options and variation in cleavage around residues 88 - 92 of basic protein presumably result from pH-dependent changes in conformation in the is region but could also be due to changes in conformation of cathepsin D. These results indicate that local tissue changes such a pH amy affect not only the velocity of the reaction but also the nature of th product formed by the degradation of basic protein by brain cathepsin D     

A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins

The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6 The dodecyl sulfate-induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate: protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1–115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116–169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2 Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: (a) that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and (b) that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent

The localization of proteins in myelin was studied by the use of a non-penetrating penetrating reagent. Tritiated 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with ³H-labelled, 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

METABOLISM OF HIGHLY BASIC PROTEINS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— (1) The encephalitogenic basic protein obtained from adult rat brain by treatment with 0·03 N-HCl was demonstrable in the brain on the 10th day after birth. It showed a marked increase in quantity during the phase of active myelination.
(2) The proteins extracted under similar conditions from 5-day old rat brain contained several highly basic proteins other than the encephalitogenic basic protein. These basic proteins, which were electrophoretically similar to highly basic proteins extracted similarly from adult rat liver, are histones.
(3) For metabolic studies the entire group of highly basic proteins in the acid extract was obtained after one-step adsorption of other proteins on DEAE-cellulose equilibrated at pH 9·8
(4) After injection of [¹⁴C]lysine the fractions containing highly basic proteins, water soluble non-basic proteins and other tissue proteins of the brain showed higher relative specific radioactivities during the period 1–10 days after birth than during later stages of postnatal development. The fraction containing proteolipid protein, another myelin protein, showed a low relative specific radioactivity throughout the whole period of postnatal development. The relative specific radioactivity of proteolipid protein was somewhat higher in young than in adult rat brain.     

Cross-linking studies on the conformation and dimerization of myelin basic protein in solution.

Myelin basic protein was isolated from both cat and bovine central nervous system. Cat and bovine myelin basic protein, which are shown to be similar by tryptic mapping, exhibit identical behavior when cross-linked with the bifunctional reagent difluorodinitrobenzene. Myelin basic protein is cross-linked into only a dimer under certain conditions in the presence of sodium dodecyl sulfate. In contrast, many oligomers are formed when myelin basic protein is cross-linked in the absence of detergent. The formation of cross-linked dimers in the absence of other oligomer formation suggests that the protein is at least partly dimeric in the presence of sodium dodecyl sulfate. The conformation of them myelin basic protein monomer in sodium dodecyl sulfate was also studied. N-Bromosuccinimide and cyanogen bromide cleavage reactions were used to demonstrate that difluorodinitrobenzene had introduced intramolecular cross-links between the two peptides resulting from each of the cleavage ractions. However, these types of intramolecular cross-links cannot be detected under conditions in which only dimers have formed. Some of the lysine residues which are modified by difluorodinitrobenzene were identified by tryptic mapping. In several respects, the conformation of myelin basic protein in a sodium dodecyl sulfate solution appears to be similar to the conformation of the protein in the membrane.     

Effect of chemical modifications of myelin basic protein on its interaction with lipid interfaces and cell fusion ability

Summary The ability of native and chemically modified myelin basic protein to induce fusion of chicken erythrocytes and to interact with lipids in monolayers at the air-water interface and liposomes was studied. Chemical modifications of myelin basic protein were performed by acetylation and succinylation: the positive charges of the native protein were blocked to an extent of about 90–95%.Cellular aggregation and fusion of erythrocytes into multinucleated cells was induced by the native myelin basic protein. This effect was diminished for both acetylated and succinylated myelin basic protein. Native myelin basic protein penetrated appreciably in sulphatide-containing lipid monolayers while lower penetration occurred in monolayers of neutral lipids. Contrary to this, both chemically modified myelin basic proteins did not show any selectivity to penetrate into interfaces of neutral or negatively charged lipids. The intrinsic fluorescence of the native and chemically modified myelin basic proteins upon interacting with liposomes constituted by dipalmitoylphosphatidycholine, glycosphingolipids, egg phosphatidic acid or dipalmitoylphosphatidyl glycerol was studied. The interaction with liposomes of anionic lipids is accompanied by a blue shift of the maximum of the native protein emission fluorescence spectrum from 346 nm to 335 nm; no shift was observed with liposomes containing neutral lipids. The acetylated and succinylated myelin basic proteins did not show changes of their emission spectra upon interacting with any of the lipids studied. The results obtained in monolayers and the fluorescence shifts indicate a lack of correlation between the ability of the modified proteins to penetrate lipid interfaces and the microenvironment sensed by the tryptophan-containing domain.Abbreviations MBP myelin basic protein - DPPC dipalmitoyl phosphatidylcholine - DPPG dipalmitoyl phosphatidylglycerol - PA phosphatidic acid     

ISOLATION OF PURIFIED BASIC PROTEIN FROM HUMAN BRAIN

—A simplified method for the isolation of an encephalitogenic basic protein from myelin is described. The basic polypeptide is easily extracted from the interfacial protein which separates when chloroform-methanol extracts of myelin are treated with a synthetic upper phase containing citrate. Evidence is presented for the purity and identity of the protein. The disc polyacrylamide gel electrophoretic pattern of this protein was consistent with a high degree of homogeneity.     

A RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN AND ITS USE FOR QUANTITATIVE MEASUREMENTS

—A specific radioimmunoassay (RIA) for myelin basic protein is described which is sensitive to 10⁻⁹ g of basic protein. The amount of basic protein detected in isolated myelin by the RIA and by SDS-gel electrophoresis and spectrophotometric quantitation agree to within experimental error. In contrast to isolated myelin, the major portion of the basic protein in fresh tissue is not accessible to its antibody. It is shielded from its antibody in a complex which is disrupted by heat, organic solvents, and various detergents. Maximum antibody binding was obtained with tissue heated to 100°C for 10 min. It is possible to calculate that the RIA quantitatively detects basic protein in boiled tissue. Boiled adult rat brain contains approximately 2·5 μg of basic protein/mg wet wt of cerebral cortex. The antibody to basic protein has no capacity to bind non-neural tissues.     

Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis

The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.