REGULATION OF THE LEVEL OF ENDOGENOUS PHOSPHORYLATION OF SPECIFIC BRAIN PROTEINS BY DIPHENYLHYDANTOIN1

Diphenylhydantoin (DPH) in therapeutic concentrations caused a decrease in the net level of endogenous phosphorylation of two specific proteins from rat brain cerebri, while not significantly affecting the phosphorylation of other protein substrates. The apparent molecular weights of the DPH- specific substrate proteins were 60-63,000 and 49-52,000, and were designated proteins DPH-L and DPH-M, respectively. DPH decreased both the initial rate and the net level of [³²P] phosphate incorporation from [γ-³²P] ATP into proteins DPH-L and DPH-M. The concentrations of DPH required to produce a half maximal decrease in the levels of phosphorylation of proteins DPH-L and DPH-M was 3 × 10^-4 and 8 × 10 ^-4 M, respectively. The effects of DPH on the incorporation of [³²P] phosphate into these specific brain proteins were independent of the concentration of ATP over a wide range of ATP concentrations. The DPH-specific proteins were demonstrated to be present in synaptosomal preparations. The results are compatible with the hypothesis that some of the stabilizing actions of DPH on neuronal tissue and seizure discharge may be mediated by the effect of this anticonvulsant on the phosphorylation of specific brain protein substrates.     

ISOLATION OF TWO PANTOTHEINE-CONTAINING ACIDIC PROTEINS FROM MONKEY BRAIN

The isolation of two acid-soluble proteins from Macaca irus brain is reported. The proteins contained 34 and 35.6 per cent polar amino acids, respectively. One protein had a mol. wt. of approx. 37,100 and the other of 23,000 with subunits of 11,500. Each protein contained 4′-phosphopantetheine, which was hydrolytically determined as taurine and β-alanine and microbiologically as pantothenate. The smaller protein is found to be present in the supernatant following homogenization both in 0-32 M-sucrose and in 0-32 M-sucrose with 40 mm -NaCl. The larger protein is also mainly present in the supernatant fluid. The function of these proteins is unknown, but the mol. wt. 23,000 species could be acetylated from glucose and acetate in mouse brain tissue slices and the acetyl groups could be isolated as the hydroxamate.     

THE NATURE OF THE IONIZABLE GROUPS IN PROTEINS

Analysis of the experimental titration curves shows that gelatin contains acid groups with dissociation indices at pH 2.9 to 3.5 corresponding quantitatively with the content in dicarboxylic amino acids; and that the acidic group at pH 9.4 in egg albumin agrees with the amount of tyrosine. The amounts of histidine and lysine present in both these proteins agree quantitatively with basic groups at pH 6.1 and pH 10.4 to 10.6, respectively. However, the quantity of the arginine group (pH 8.1) in these proteins is considerably less than the amount of arginine found on hydrolysis. This deficiency is compensated (quantitatively with gelatin and approximately with egg albumin) by a basic group at pH 4.6. The structure of this "4.6 group" should be similar to aniline and cytosine in consisting of an amino group on a conjugated unsaturated (perhaps cyclic) system. It would appear that the 4.6 group is disrupted on hydrolysis, producing arginine, and may be referred to as "prearginine." The presence of prearginine in proteins, instead of the full amount of arginine, has an important effect on the properties. Otherwise the isoelectric point of gelatin would be 8.0 (instead of 4.7) and of egg albumin 6.6 (instead of 4.8), and the titration curves would be quite different in shape between pH 4 and 10. Deamination of gelatin produces no decrease in prearginine, arginine, or histidine groups, but removes nearly all of the lysine group.     

NUCLEAR PROTEINS OF THE GUINEA PIG BRAIN

Abstract—

• 1 Chromatin protein fractions were separated from the nuclei from brain, liver and kidney of the guinea pig. The fractions were studied by electrophoretic methods and amino acid analysis.
• 2 Brain nuclear fractions were washed with 0.15m -NaCl and nuclear acidic proteins then removed by 0.35 m -NaCl. These 0.35 m -NaCl-extracted proteins were considered to be similar to the nuclear soluble acidic proteins.
• 3 Nonhistone-1, histone and nonhistone-2 fractions were obtained from 2.0 m -NaCl-soluble chromatin fractions by lowering the salt concentration and successive extraction with acid and alkali. The nonhistone-3 fraction was also extracted from the nuclear residue by alkaline solution.
• 4 The contents and characteristics of the nonhistone fractions of the brain, especially the nonhistone-1 fraction, differed among the three tissues. The histone fractions showed no obvious difference among the three tissues. The nonhistone-1 fraction of the brain, which comprised a low percentage of total nuclear protein, contained relatively high amounts of acidic proteins.

     

兰州百合精细胞特异蛋白的研究

通过低渗冲击及Percoll密度梯度离心的方法,成功地分离并纯化了兰州百合(Lilium davidiiDuch.)生活的生殖细胞及精细胞。从精细胞、生殖细胞及叶片中提取了全蛋白,并通过双向电泳技术对它们进行了比较。在双向电泳图谱上精细胞比生殖细胞显示更多的蛋白斑点,特别是在碱性端。通过混合酶解及离心,分离了生活的叶肉原生质体。用生物素的琥珀酰胺酯衍生物(NHS-biotin)对精细胞、生殖细胞及完整的叶肉原生质体质膜蛋白进行标记,然后进行Western blot分析,用辣根过氧化物酶酶标链霉抗生物素蛋白及其底物4-氯-1-萘酚反应显色,比较了3种质膜蛋白。发现分子量为46kD及50kD的两种蛋白是精细胞质膜特异的。在双向电泳图谱上也可找到与这两种蛋白相对应的斑点,它们很可能与受精过程中精卵的识别有关。     

RAPIDLY LABELED AND SECRETED PROTEINS OF THE CHICK BRAIN

Abstract— The extracellular and cerebrospinal fluids (ECF) of the chick brain were found to contain a distinctive group of rapidly labeled proteins. Gel staining patterns suggest that most ECF protein bands correspond with components also found in either the homogenized whole brain cytoplasmic fraction or the blood serum. The valine-incorporation profiles of these three fractions, however, were entirely distinctive. Comparisons were carried out using a sensitive double-labeling method, in which ECF proteins from chicks labeled for 1 h with [³H]valine were comigrated on SDS-polyacrylamide gels with the cytoplasmic or serum proteins from a ¹⁴C-labeled animal. Analyses of the ³H- and ¹⁴C-labeling profiles from these gels showed that certain newly-synthesized proteins are heavily enriched in the ECF relative to the other two fractions. Most prominently, material with an apparent molecular weight of # 17,000 was found to incorporate nearly one-third of all the radioactivity appearing in the ECF proteins, but was not heavily labeled in either the cytoplasmic or serum fractions. The effects of a simple training experience on the pattern of chicks' brain protein synthesis were also examined.     

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.     

THE NATURE OF THE INCREASED RATE OF DNA SYNTHESIS IN SCRAPIE-AFFECTED MOUSE BRAIN

—In vivo studies have been made of the incorporation of radioactive thymidine into acid insoluble components of control and scrapie-affected mouse brain. Experiments with hot trichloroacetic acid extracts of brain and with purified preparations of DNA have confirmed that there is an increased rate of DNA synthesis in scrapie brain which is entirely associated with nuclei. No increase was found in the rate of DNA synthesis in cytoplasmic fractions of scrapie brain. Hydroxyapatite chromatography of heat denatured and renatured DNA suggests that in scrapie brain there is a similar increase in the rates of synthesis of the poorly, moderately and highly reiterated (i.e. satellite) species of nuclear DNA. Experiments involving brain dissection indicate that the increased rate of DNA synthesis in scrapie does not take place exclusively in the subependymal layer of the lateral ventricles. From these and previously reported studies using radioautographic techniques it is concluded that the increased DNA synthesis in scrapie brain is not associated specifically with cells undergoing mitosis.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

玉米精细胞质膜特异蛋白的纯化

在获得6个品种的玉米(Zea mays L.)花粉精细胞后,采用N-hydroxysuccinimido-biotin(NHS-biotin)标记其外膜蛋白,并通过SDS-PAGE和Western blotting对比了其中主要标记蛋白,发现其主要蛋白带差异并不显著,主要标记蛋白分子量均集中于91、60、43、30和17kD。采用免疫亲和层析技术进一步纯化已获得的混杂少量其它细胞器成分的精细胞质膜制剂,即利用制备的体细胞主要细胞器:线粒体、内质网、高尔基体及质膜的膜蛋白,分别免疫豚鼠,从其抗血清中纯化获得IgG,并进一步制成各种膜蛋白的免疫亲和吸附制剂。利用此技术进一步纯化经NHS-biotin标记的精细胞质膜蛋白,获得精细胞质膜特异的蛋白质,其中最为显著的蛋白质分子量约为65、22kD。     

THE NATURE OF SULPHATION OF URONIC ACID-CONTAINING GLYCOSAMINOGLYCANS CATALYSED BY BRAIN SULPHOTRANSFERASE

—A sulphotransferase system of rat brain catalyses the transfer of sulphate from 3′-phosphoadenosine 5′-phosphosulphate to the low-sulphated glycosaminoglycans isolated from normal adult human brain. These were shown to be precursors of higher-sulphated glycosaminoglycans by DEAE-Sephadex column chromatography and paper electrophoresis. Nitrous acid degradation and mild acid hydrolysis of enzymically-sulphated fractions further confirmed the presence of heparan sulphate in human brain. A partially purified sulphotransferase preparation was obtained from neonatal human brain using chondroitin-4-sulphate as sulphate acceptor. This sulphotransferase catalyses the transfer of sulphate to the various uronic acid containing glycosaminoglycans. Heparan sulphate was the best sulphate acceptor followed by dermatan sulphate, N-desulphoheparin, chondroitin-4-sulphate and chondroitin-6-sulphate in decreasing order. Sulphotransferase obtained from 1-day-old rat, rabbit and guinea pig brain also had the same pattern of specificity towards various sulphate acceptors. This sulphotransferase catalyses both N-sulphation and O-sulphation. Studies on the sulphotransferase obtained from both rat and human brain of various age groups indicate that the ratio of N-sulphation: O-sulphation decreases as the brain matures.     

WALLERIAN DEGENERATION IN RABBIT OPTIC NERVE: CELLULAR LOCALIZATION IN THE CENTRAL NERVOUS SYSTEM OF THE S-100 AND 14-3-2 PROTEINS1

Abstract— The S-100 and 14-3-2 proteins, which are found only in nervous tissues, were measured in degenerating rabbit optic nerve at 0, 5 10, 20, 40, 60, 80, 100, 150 and 200 days after unilateral enucleation in order to obtain indications of the cellular localization of these proteins in the central nervous system. S-100 increased and 14-3-2 decreased (both approximately 70 per cent) in cut nerves by 200 days of degeneration. Changes in amounts of the proteins were related to cellular alterations which characterize the degenerative process, as demonstrated by electron microscopy. In uncut nerves (intact eye) from these experimental animals, S-100 increased and 14-3-2 decreased slightly at 5 days, after which time the levels of each returned to those approximating the content in corresponding nerves from unoperated control animals. No appreciable change in total soluble proteins was measured in degenerating or intact nerves. Since S-100 increased and 14-3-2 decreased in the degenerating optic nerve as it became relatively enriched in glial constituents but impoverished in axonal content, it is suggested that S-100 is primarily a glial protein and 14-3-2 predominantly a neuronal protein in the central nervous system.     

IMMUNOCHEMICAL STUDIES ON THE BRAIN SPECIFIC PROTEIN

Abstract— In the soluble brain proteins of various species-man, ox, cat, rabbit, rat, mouse, hen, snake, frog and fish–there is a protein group which migrates more slowly than Moore's S-100 protein and faster than the albumin fraction on disc electrophoresis. The protein group is absent from any organs other than brain, and has a different pattern and number of fractions in different species. Immunochemically, the protein fraction group of the mammalian brains shows some common and identical distinctive antigenic determinants compared with the brain protein of the other animals-hen, snake, frog and fish. The protein group was designated the 'SPR' proteins, which were separated to 'P_II,', 'P_III', 'P_IV' and 'P_v' fractions. Common antigenic determinants are found in these fractions. The protein group is found in human brain in larger amounts in grey matter than in white matter and in small amounts in the cellular nuclei of human and bovine brain.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.