DISTRIBUTION AND OPTICAL ACTIVITY OF THE BASIC PROTEIN IN BOVINE PERIPHERAL NERVE MYELIN

Abstract— Antiserum to BF protein isolated from bovine spinal roots has been used to study the distribution of the protein in other species and tissues.
Significant amounts of protein could be demonstrated in bovine, pig and rabbit peripheral nerve myelin. It was, however, scarcely detectable in guinea pig peripheral nerve myelin. There was BF protein in rabbit spinal cord as well as in peripheral nerve, but little or no BF protein in the liver, kidney, muscle or brain. BF protein in bovine spinal cord was localized in the myelin. The ratio of the BF protein to the encephalitogenic protein in the spinal cord myelin was around 0.15:1.0. BF protein was extractable from peripheral nerve myelin by saline as well as by acid solutions.
The circular dichroism spectrum of the BF protein in aqueous solution suggested that this protein contained a very large amount of β-structure. This structure was not considered to be the result of acid denaturation because the protein purified from the saline extract of peripheral nerve also showed a similar spectrum.     

STRUCTURAL AND IMMUNOLOGICAL PROPERTIES OF NEURON SPECIFIC PROTEIN (NSP) FROM RAT, CAT AND HUMAN BRAIN: COMPARISON TO BOVINE 14-3-2

Abstract— Neuron specific protein (NSP) has been isolated from cat (NSP-C) and human (NSP-H) brain utilizing the purification procedure described for rat brain 14-3-2 (M arangos et al. , 1975a,b,c), a protein which is now designated NSP-R. The protein as isolated from cat and human brain has a molecular weight of approx 80,000 as determined by sedimentation equilibrium. Sedimentation studies done in the presence of 6mg-HCl and 0.2%β mercaptoethanol yields a protomer M.W. of approx 40,000 for both preparations establishing the dimeric nature of each. The subunits appear identical in each case since one band is observed upon electrophoresis of either preparation in the presence of 8 M-urea. NSP-C and NSP-H have identical isoelectric points of 4.7 making them slightly more acidic than NSP-R (pi = 5.0).
Comparison of NSP-C and NSP-H with NSP-R and bovine 14-3-2 by electrophoretic and immunological criteria revealed that the cat, human and bovine proteins were very similar. NSP-R can be distinguished from the other three preparations electrophoretically and immunologically. The protomer unit of NSP-R differs in amino acid composition from that of the cat, human or bovine proteins since the former can be completely resolved from any of the latter three preparations on 8 M-urea polyacrylamide gels. The data indicate that NSP and bovine 14-3-2 are probably homologous proteins, and establish the general structural properties of NSP.     

COMPARATIVE STUDIES OF HIGHLY BASIC PROTEINS OF OX BRAIN AND RAT BRAIN. MICROHETEROGENEITY OF BASIC ENCEPHALITOGENIC (MYELIN) PROTEIN

(1) The total amount of highly basic proteins in acid extracts of whole ox brain, ox white matter and ox grey matter was determined quantitatively after electrophoresis on 5% polyacrylamide gels at pH 10-6 in the presence of 8 M-urea. (2) Ox white matter gave 13 mg and ox grey matter 2 mg of highly basic proteins per g fresh tissue on treatment with 0-03 n -HCl. The yield of total basic proteins of ox white matter increased to 17-6 mg/g fresh brain on stepwise extraction at pH 3-0, 2-0 and 1-0; the extract at pH 3.0 accounted for 90 per cent of the total basic proteins. (3) The high encephalitogenic activity of the fraction of highly basic proteins extracted at pH 3.0 from ox white matter indicated that these basic proteins were derived from myelin. It is suggested that the amount of basic proteins in a sample of brain extracted under these conditions is proportional to the amount of white matter in the sample. (4) The encephalitogenic (myelin) basic protein fraction was homogeneous with respect to molecular size but could be resolved into at least six components by electrophoresis at high pH. (5) The myelin basic proteins extracted from ox white matter had lower electrophoretic mobilities at high pH than did those of two basic proteins of rat brain apparently derived from myelin.     

COMPARATIVE STUDIES ON THE MYELIN PROTEINS OF BOVINE PERIPHERAL NERVE AND SPINAL CORD

Abstract— (1) Two myelin fractions of bovine peripheral nerve and spinal cord have been studied comparatively. Cholesterol as well as cerebroside content per mg of protein in the peripheral nerve myelin was less than that in the spinal cord myelin, while no significant difference in the total phospholipid content was noted.
(2) The basic proteins in myelin fractions were quantitatively estimated by disc gel electrophoresis. Around one-fourth of the total myelin protein in the bovine peripheral nerve was a basic protein with a mobility of 1.07 relative to lysozyme by Reisfeld's disc gel electrophoresis.
(3) The myelin proteins in the peripheral nerve were less completely solubilized than those of the spinal cord by treatment with deoxycholate as well as by Triton-salt solution. The protein fractions obtained from the peripheral nerve myelin by techniques similar to that for obtaining the proteolipids from the spinal cord myelin, contained different types of protein.
(4) 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the peripheral nerve myelin was only one tenth of that in the spinal cord myelin. The Triton-salt insoluble fraction showed remarkable high activity among subfractions of the spinal cord myelin.
(5) By immunological studies, it may be concluded that an antigenic substance for experimental allergic neuritis was localized in the peripheral nerve myelin, but not in its basic protein.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

PROTEINS AND GLYCOPROTEINS IN MYELIN PURIFIED FROM THE DEVELOPING BOVINE AND HUMAN CENTRAL NERVOUS SYSTEMS

Myclin was purified from bovine and human midbrain at various stages of prenatal and postnatal development. Basic protein and proteolipid proteins were the major individual proteins at all stages. The specific activity of 2′3′-cyclic nucleotide 3′-phosphohydrolase remained constant in the bovine myclin during development but decreased slightly in human myelin. A high molecular weight glycoprotein with electrophoretic mobility similar to that previously reported in rodent myelin (QUARLES et al., 1973) was present in both bovine and human myelin at all stages of development. The intensity of staining of this glycoprotein with periodic acid-Schiff reagents per mg total myelin protein was less in mature bovine and human myelin than in rat myelin.     

MICROHETEROGENEITY AND PHOSPHOAMINO ACIDS IN THE CARBOXY-TERMINAL HALF OF MYELIN BASIC PROTEIN

—Three chromatographically distinct peptic peptides (F80-1, F80-2 and F80-3) derived from the C-terminal half of the bovine and guinea-pig myelin basic proteins were characterized. The three peptides of each animal species had the same N-terminal residue (phenylalanine) and essentially the same amino acid composition, but they differed in electrophoretic mobility at alkaline pH. The least basic peptide (F80-3) differed from the others in showing a deficit of C-terminal arginine residues and in containing phosphorus, 0·37 and 0·46 g-atom/mol of bovine and guinea-pig peptides, respectively. Other peptic peptides. derived from the N-terminal half of the basic protein, were essentially phosphorus-frcc. Analyses of partial acid hydrolyzates of peptide F80-3 by high voltage electrophoresis showed the presence of both phosphoserine and phosphothreonine. After incubation with E. coli alkaline phosphatase (EC 3.1.3.1). 34 and 40% of the bovine and guinea-pig F80-3 peptides. respectively, were converted to peptide F80-1. This reaction involved the loss of 2 net negative charges, and its extent corresponded to loss of essentially all of the phosphate originally present in the peptide. This result indicated that the phosphorylated species of peptide F80-3 contained one phosphate group per molecule.     

PROTEIN AND GLYCOPROTEIN COMPOSITION OF MYELIN AND MYELIN SUBFRACTIONS FROM BRAINS OF ''QUAKING'' MICE

Abstract— Quaking mutants in mice are known to be affected by an arrest of myelinogenesis and to have a purified myelin which is more dense than that of controls. Their myelin has been shown to demonstrate a striking decrease in proteolipid protein, a lesser decrease in the small myelin basic protein and changes in glycoproteins comprising reduction in the major peak and shift of this peak towards a higher apparent molecular weight. The possibility that these findings might reflect merely contamination of myelin with other membranes was tested by subfractionation. Light myelin (floats on 0.62 m -sucrose) is generally accepted as more compact and mature than the heavier subfraction (floating on 0.85 m -sucrose). The changes previously found were present in both subfractions and even more marked in the light myelin. These results indicate that the anomalies of myelin proteins and glycoproteins were not caused by contaminants and are present in compact myelin as well as in membranes which are transitional between the glial plasma membrane and the myelin sheath. Therefore, we suggest that the Quaking mutation results in dysmyelination rather than hypomyelination.     

COMPARATIVE STUDIES OF GUINEA PIG AND BOVINE MYELIN BASIC PROTEINS. PARTIAL CHARACTERIZATION OF CHEMICALLY DERIVED FRAGMENTS AND THEIR ENCEPHALITOGENIC ACTIVITIES IN LEWIS RATS

Guinea pig and bovine myelin basic proteins were chemically cleaved at the carboxyl peptide bonds of methionyl and tryptophanyl residues to yield several fragments. Comparison of the bovine fragment consisting of the first 20 residues of the protein with the corresponding guinea pig fragment showed that the latter differs in containing histidine and glycine (one residue of each), an additional threonyl residue, and one fewer alanyl residues. Comparison of the bovine fragment consisting of the C-terminal 54 residues of the protein (residues 117-170) with the corresponding guinea pig fragment showed that the latter differs in containing one fewer histidyl and leucyl residues and an additional phenylalanyl residue. Tests of encephalitogenic activity in Lewis rats showed that these two fragments from both species were much less active, on a molar basis, than the uncleaved protein. On the other hand, examination of the bovine fragments consisting of residues 1-116 and 21-116 and the corresponding fragments obtained from the guinea pig protein revealed activity at least as high as that of the respective uncleaved proteins.     

PURIFICATION AND SOME PROPERTIES OF CHOLINE ACETYLTRANSFERASE (EC 2.3.1.6) FROM BOVINE BRAIN

Abstract— Choline acetyltransferase (ChAc) has been isolated and highly purified from the caudate nuclei of bovine brains. The procedure involved: (1) making an acetone- and chloroform-insoluble powder from the tissue; (2) treating the powder with aqueous buffer and chromatographing the extractable soluble proteins on an organomercurial-sepharose column; (3) removing impurities by passage through columns of DEAE-cellulose and hydroxyapatite; and (4) separation of the heme-containing protein from ChAc by denaturing the former with a mixture of chloroform and n -butanol. The purified ChAc was essentially homogeneous as judged by polyacrylamide gel electrophoresis and exhibited a pH optimum at about pH 7. The partially purified ChAc dissociated into two non-identical subunits when chromatographed with a dilute buffer on Bio-gel A. It did not dissociate when a more concentrated buffer was used. The purified ChAc dissociated on the Bio-gel A even in the presence of a high salt concentration. The dissociation was accompanied by a great loss of enzymatic activity, and we concluded that the presence of other proteins tends to prevent the dissociation of ChAc on gel filtration.     

FURTHER CHARACTERIZATION OF BOVINE KERATOHYALIN

Extraction of serial sections of cattle hoof epidermis with solutions of calcium chloride, magnesium chloride, potassium chloride, sodium chloride, guanidine hydrochloride, ammonium sulfate, and potassium phosphate buffer (pH 7.0) at varying salt concentrations demonstrates that keratohyalin (KH) is extracted by these salts at certain molarities. Under given conditions of time and temperature, each salt has a specific extraction pattern, and similar salts have similar extraction patterns. Dialysis of the salt extracts of hoof epidermis against distilled water results in the macroaggregition of KH, as assayed by histochemical methods. Although the various macroaggregates appear identical at the histochemical level, they display different ultrastructural characteristics. Polyacrylamide gel electrophoresis of the sodium decyl sulfate-solubilized macroaggregates results in the fractionation of a 20 (or more) member homologous series of oligomers. Isolation of the various oligomeric species of bovine keratohyalin and re-electrophoresis indicate that the various KH species can undergo depolymerization. Amino acid analyses of the unfractionated bovine macroaggregates and the various molecular weight species of bovine KH are similar, further demonstrating homology of the oligomers. The molecular weight of the subunit (monomer) of bovine KH is 14,955, estimated from the amino acid analyses.     

曲霉和青霉属内杂种和亲株的蛋白质电泳图谱比较

曲霉属内黑曲霉(Aspergitlus niger)与米曲霉(A.oryzae)具有特征明显不同的可溶性蛋白质电泳图谱,其种间杂种具有双亲的部分或全部电泳带并与黑曲霉相近。来自杂种Ⅰ的多数分离子电泳带与黑曲霉相近,只有一个分离子产生米曲霉的电泳带并具有米曲霉的遗传特性。青霉属内产黄青霉(Penicillium chrysogenum)与展青霉(P.patulum)种间及种内不同菌株间的电泳图谱基本相同,种内或种间杂种具有双亲的电泳带。结果讨论了蛋白质图谱分析的意义。     

PROTEIN PATTERNS IN DIFFERENT LOBES AND DURING DEVELOPMENT OF OCTOPUS BRAIN

Abstract— Utilizing techniques of continuous and SDS-electrophoresis we have examined the saline-soluble and SDS-soluble (membrane-bound) proteins extracted from the main lobes of adult octopus brain and from the developing optic lobe of the same species. Several additional protein bands are present among the soluble and the membrane-bound proteins of the vertical lobe in comparison with the suboesophageal lobe. Since the former contains an essentially homogeneous population of small neurons, while the suboesophageal lobe is rich in large nerve cells, these protein bands have been attributed to the small neuronal type present in the vertical lobe.
In the course of a 10,000-fold increment in body weight, from 0.4 g to 4 kg, there is a significant increase in the concentration of several soluble proteins extracted from the optic lobe. Three of these proteins increase to a marked degree. Among the membrane-bound proteins some show a moderate increase with age while other protein components of smaller molecular weight undergo a moderate decrease. The overall tissue concentration of the membrane-bound proteins increases between 0.4 g and 50 g body weight, slightly declining in animals of larger size.     

ON THE SEQUENTIAL CLEAVAGE OF MYELIN BASIC PROTEIN BY CATHEPSINS A AND D

Abstract— A lysosomal carboxypeptidase (cathepsin A) known to increase in demyelination (experimental allergic encephalomyelitis and multiple sclerosis) and purified 277 fold from bovine brain failed to attack myelin basic protein. Prior treatment of basic protein with bovine cathepsin D (EC 3.4.23.5) at pH 3.2 led to the production of three polypeptides one of which, Phe⁴³-Phe⁸⁸, acted as a substrate for the carboxypeptidase. Incubation of this fragment with cathepsin A at pH 5.5 led to the release of C-terminal Phe equivalent to 50% of the total within 6h at 25°C and smaller amounts of the adjacent His (position 87) and Val (86). Purified cathepsin A was devoid of aminopeptidase and arylamidase contaminants when tested with a variety of di- and tripeptides and with the polypeptide fragment of myelin basic protein Phe⁸⁹-Arg¹⁶⁹. These studies in vitro with two different lysosomal enzymes show that cathepsin D activity is rate-limiting. A demonstration of step-wise cleavage by lysosomal enzymes may be relevant to mechanisms regulating protein turnover and to processes involved in demyelination.     

Binding of protein SCP to myelin, its identity with protein P2. A simple method of protein P2 isolation

Protein SCP is found in myelin of spinal cord and spinal roots. It is shown that its amount accounts for 12% of the total protein content in myelin of spinal roots and only for 2% in myelin of spinal cord. Almost all the studied protein is extracted from myelin with 0.1 M NaCl (80-90%). The absolute identity of protens SCP and P2 is established using the cross reaction immunodiffusion with monospecific antisera. It is shown that- N-terminal amino acid in protein SCP, like in protein P2 is blocked. On the basis of the data obtained a conclusion is made that protein P2 is not an integral protein of myelin. However, myelin is capable under conditions of a nonionic medium of binding protein which then may be easily extracted by increasing the medium ionic strength. This gave reasons to propose a method for protein P2 isolation from myelin using 0.15 M NaCl with the subsequent purification by means of Sephadex G-50 gelfiltration.     

QUAKING MOUSE: ISOLATION AND CHARACTERIZATION OF MYELIN PROTEIN

A new technique, involving final purification on a continuous CsCl gradient, was utilized for the isolation of cerebral myelin from adult (4- to 6-month-old) quaking mice, littermate controls and young (10-day-old) normal mice. The yield of myelin from either adult quaking or normal young mice was 5-10 per cent of that from adult controls. After deli-pidation, myelin proteins were separated by polyacrylamide gel electrophoresis in buffers containing sodium dodecylsulphate. Two gel systems were utilized: (1) a high-resolution discontinuous electrophoresis system; and (2) a continuous system utilizing gels cross linked with ethylenediacrylate (EDA). The gels from the discontinuous system were stained with Fast Green and quantified by densitometry. The base lability of the EDA-linked gels permitted direct chemical determination of protein in specific bands. Myelin from brains of normal adult mice contained, as major components, one proteo-lipid and two basic proteins. There were also a number of high-molecular-weight proteins which represented a significant portion of the total. Myelin from quaking mice had qualitatively a similar distribution of proteins but the high-molecular-weight fraction comprised a much greater percentage of the total protein. The ratio of basic to proteolipid protein in preparations from quaking mice was considerably higher than that in the myelin from control mice. The distribution pattern of the myelin proteins from 10-day-old mice was quantitatively similar to that of quaking mice. Altogether the evidence supports the hypothesis that the quaking mutant provides a model of an immature nervous system with respect to myelination.     

On basic proteins in bovine peripheral nerve myelin.

1. Myeline proteins in bovine peripheral nerve migrated as two main band-(BF and BR protein) and one faint middle band (BM protein) on sodium dodecyls sulfate-polyacrylamide gel electrophoresis. The relative mobility of these two main bands differed from those of myelin proteins in the central nervous system. 2. The acid extract of the myelin fraction from bovine peripheral nerve was separated into one main peak and two minor peaks on a Sephadex G-75 column. The major component of the second minor peak was the BM protein; the major component of the main peak was the BF protein. The BR protein was not extractable by acid solution. 3. Molecular weights of the BF, the BM and the BR protein were determined as around 13 000, 20 000 and 28 000, respectively, by sodium dodecylsulfate-polyacrylamide gel electrophoresis. 4. The amino acid composition of the BF protein was quite different from the encephalitogenic protein and the Folch-Lees type proteolipid protein in the central nervous system. However the BM protein showed similar amino acid composition to the encephalitogenic protein. 5. The tryptic peptide maps of the BF protein and of the encephalitogenic protein were quite different. The results suggested that the amino acid sequences of these two proteins are different and that they contain no common tryptophan-containing peptide.     

Further characterization of the anti-encephalitogenic protein (SCP): isolation from bovine spinal cord and spinal roots.

The three molecular forms of the anti-encephalitogenic protein, beta-SCP, gamma-SCP, and SCP-peptide were isolated in higher yield by a shortened procedure, which involved 1) extraction of bovine spinal cord (BSC) or bovine spinal roots (BSR) with 0.05 M sodium acetate buffer, pH 4.5, 2) batch absorption on CM-52 cellulose, 3) stepwise elution with sodium acetate buffers, pH 5.8, containing increasing concentrations of sodium chloride and finally, 4) removal of trace contaminants by gel-exclusion chromatography on Sephadex G-50 superfine. The m.w. of the purified proteins determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was 13,200 daltons. The same value for the molecular sizes was obtained by gel-exclusion chromatography by using 0.1% SDS in 0.05M sodium chloride as eluant. In the absence of SDS the molecular sizes estimated by gel exclusion chromatography ranged from 14,000 to 18,500. The amino acid compositions of the beta-SCP and gamma-SCP from BSC and BSR were similar except that beta-SCP from BSR lacked half-cystine whereas gamma-SCP from BSR contained three times as much half-cystine as the SCP forms prepared from BSC. All forms of SCP showed reactions of identity when compared by immunodiffusion analyses with a rabbit anti-bovine SCP serum; none formed precipitin lines with a rabbit anti-bovine myelin basic protein (MyBP) serum.     

THE ISOLATION AND PROPERTIES OF LARGE BASIC PEPTIDES FROM BOVINE SPINAL CORD

Abstract— Two basic peptides (B1 and B2) were derived from bovine spinal cord following in situ proteolysis at 37°C for 10–24 h. These peptides do not arise as degradation products from the A1 protein as shown by amino acid composition and end group analysis; rather they appear to originate from some larger basic protein in the spinal cord having similarities to the P2 protein, a basic protein found in peripheral nerve myelin. The peptides were purified following defatting, acid extraction, and ammonium sulphate fractionation, by chromatography on Amberlite IRC-50 resin using guanidinium chloride. The peptides, found generally in a 4:1 ratio of B1 to B2, appeared homogeneous on gel electrophoresis and immunodiffusion. Approximately 25–60 mg of peptides was obtained per 100 g wet spinal cord.
In contrast to the basic A1 protein from myelin, neither of these peptides nor their pepsin digests were encephalitogenic. They do not cross-react immunologically with the basic A1 protein, but cross-react with each other. These peptides further differ from the A1 protein in their tryptic peptide map, size (B1, 63 residues; B2, 54 residues), and composition particularly the high lysine: arginine ratio, and low histidine content. Like the A1 protein, however, they contain a tryptophan residue and a blocked NH₂-terminal amino acid; peptide Bl has COOH-terminal valine. It was concluded that the basic peptides represent a fragment of a hitherto unidentified protein(s) of the nervous system.