Isoelectric focusing of peptides

Righetti, Pier Giorgio; Chillemi, Francesco

doi:10.1016/s0021-9673(00)92339-2

Cited by 123 publications

(34 citation statements)

References 14 publications

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“…The two procedures yield very similar peptide patterns from all of the sources examined. However, the number of peptides resolved by either technique is smaller than expected from predictions based on (i) the minimum number of CNBr peptides, calculated from protein and DNA sequence data (16-19, 59, 60), that would be large enough to detect by these procedures (57,58) and (ii) the large number of distinguishable isotubulins (Figs. 3 and 4), each of which must differ in at least one peptide.…”

Section: Methodsmentioning

confidence: 71%

Heterogeneity of vertebrate brain tubulins.

Field¹,

Collins²,

Lee³

1984

Proc. Natl. Acad. Sci. U.S.A.

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We have examined the extent of brain tubulin heterogeneity in six vertebrate species commonly used in tubulin research (rat, calf, pig, chicken, human, and lamb) using isoelectric focusing, two-dimensional electrophoresis, and peptide mapping procedures that provide higher resolution than previously available. The extent of heterogeneity is extremely similar in all of these organisms, as judged by number, range of isoelectric points, and distribution of the isotubulins. A minimum of 6 a and 12 f3 tubulins was resolved from all sources. Even the pattern of spots on two-dimensional peptide maps is remarkably similar. These similarities suggest that the populations of tubulin in all of these brains should have similar overall physical properties. It is particularly interesting that chicken, which has only four or five 8-tubulin genes, contains approximately 12 P tubulins. Thus, post-translational modification must generate at least some of the tubulin heterogeneity. Mammalian species, which contain 15-20 tubulin DNA sequences, do not show any more tubulin protein heterogeneity than does chicken. This suggests that expression of only a small number of the mammalian genes may be required to generate the observed tubulin heterogeneity.Microtubules are protein filaments composed principally of a-and f-tubulin subunits. They are involved in a variety of cellular functions including mitosis, maintenance of cell shape, cell motility, intracellular transport, and secretion (1). It has long been thought that these diverse functions are not all performed by microtubules assembled from a homogeneous pool of identical tubulin subunits (2). Indeed, there is now compelling evidence from electrophoresis (3, 4), chromatography (5, 6), isoelectric focusing (7-15), protein sequencing (16,17), and DNA sequencing (18-20) that a-and f-tubulin subunits are actually populations of heterogeneous proteins.The nature, extent, and significance of tubulin heterogeneity are not fully understood. Substantial progress has been made in determining the nature of the heterogeneity: multiple, nonidentical tubulin genes have been identified in all the higher eukaryotes that have been examined and in several, possibly all, cases more than one gene is expressed (see ref.21 for a recent review). This implies that different gene products probably account for some of the heterogeneity. Posttranslational modification may account for the remaining heterogeneity: detyrosylation (22), aminoacylation (23), phosphorylation (24), glycosylation (25,26), and acetate-metabolite addition (27) have been reported, although it is not known whether these modifications occur in all organisms. The functional significance of the heterogeneity has been more difficult to determine. Many observations suggest a correlation between tubulin differences and particular functions: expression of tubulin genes is tissue dependent (28-33); the occurrence and distribution of tubulin proteins is tissue specific (34-39); specific tubulins are associated with distinct tubulin popu...

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Section: Methodsmentioning

confidence: 71%

Heterogeneity of vertebrate brain tubulins.

Field¹,

Collins²,

Lee³

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Fractionation of the L. q. hebraeus venom was performed according to Electrophoretic Techniques SDS-PAGE and analytical isoelectric focusing were carried out as described by Swank and Munkers [19] and Righetti and Chillemi [20], respectively, and as specified in the legend to Figure 2.…”

Section: Column Chromatographymentioning

confidence: 99%

Depressant insect selective neurotoxins from scorpion venom: Chemistry, action, and gene cloning

Zlotkin

Gurevitz

Fowler

et al. 1993

Arch Insect Biochem Physiol

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The present study examines the similarity in the symptoms and binding properties between the depressant and excitatory insect-selective neurotoxins, derived from scorpion venom. A comparison of their primary structures and neuromuscular effects is presented. A new depressant toxin (LqhIT2) was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic its effects on the intact insect, i.e, a brief period of repetitive bursts of regular junction potentials (JPs) is followed by reduced amplitude JPs ending with a block of the neuromuscular transmission. "Loose" patch clamp recordings indicate that the repetitive activity has a presynaptic origin (the motor nerve) and resembles the effect of the excitatory toxin AaIT. The final synaptic block is supposed to be the end result of neuronal membrane depolarization. Such an effect is not caused by an excitatory toxin, which induces long "trains" of repetitive firing. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation indicating a high degree of sequence homology. This conservation differs from those of other groups of scorpion toxins. The opposing pharmacological effects of depressant toxins are discussed in light of the above neuromuscular effects and sequence analysis. A genetic approach in the study of the structure-function relationships of the depressant toxins was initiated by isolating cDNA clones encoding the LqhIT2 and BjIT2 toxins. Their sequence analysis revealed the precursor form of these toxins: A 21 amino acid residue signal peptide followed by a 61 amino acid region of the mature toxin, and three additional amino acids at the carboxy terminus.

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“…The apparent molecular weight of the purified Stellarmedin A was determined by using an FPLC system (TSK-Gel G4000 PWXL column) equilibrated and eluted with 50 mM phosphatebuffered saline (PBS; pH 6.8) at a flow rate of 0.8 ml/min. The isoelectric point (pI) of the purified protein was determined by isoelectric focusing under non-denaturing condition on 5% polyacrylamide gel containing ampholines (pH 8.0-10.5) using the method of Righetti and Chellemi [21].…”

Section: Purification Of Stellarmedin Amentioning

confidence: 99%

Purification and characterization of a novel anti-HSV-2 protein with antiproliferative and peroxidase activities from <italic>Stellaria media</italic>

Shan¹,

Zheng²,

Guan³

et al. 2013

ABBS

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A novel antiviral protein, designated as Stellarmedin A, was purified from Stellaria media (L.) Vill. (Caryophyllaceae) by using ammonium sulfate precipitation, cation-exchange chromatography system. Gel electrophoresis analysis showed that Stellarmedin A is a highly basic glycoprotein with a molecular weight of 35.1 kDa and an isoelectric point of ∼8.7. The Nterminal 14-amino acid sequence, MGNTGVLTGERNDR, is similar to those of other plant peroxidases. This protein inhibited herpes simplex virus type 2 (HSV-2) replication in vitro with an IC 50 of 13.18 mg/ml and a therapeutic index exceeding 75.9. It was demonstrated that Stellarmedin A affects the initial stage of HSV-2 infection and is able to inhibit the proliferation of promyelocytic leukemia HL-60 and colon carcinoma LoVo cells with an IC 50 of 9.09 and 12.32 mM, respectively. Moreover, Stellarmedin A has a peroxidase activity of 36.6 mmol/min/mg protein, when guaiacol was used as substrate. To our knowledge, this is the first report about an anti-HSV-2 protein with antiproliferative and peroxidase activities from S. media.

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Isoelectric focusing of peptides

Cited by 123 publications

References 14 publications

Heterogeneity of vertebrate brain tubulins.

Heterogeneity of vertebrate brain tubulins.

Depressant insect selective neurotoxins from scorpion venom: Chemistry, action, and gene cloning

Purification and characterization of a novel anti-HSV-2 protein with antiproliferative and peroxidase activities from <italic>Stellaria media</italic>

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Isoelectric focusing of peptides

Cited by 123 publications

References 14 publications

Heterogeneity of vertebrate brain tubulins.

Heterogeneity of vertebrate brain tubulins.

Depressant insect selective neurotoxins from scorpion venom: Chemistry, action, and gene cloning

Purification and characterization of a novel anti-HSV-2 protein with antiproliferative and peroxidase activities from &lt;italic&gt;Stellaria media&lt;/italic&gt;

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Product

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Purification and characterization of a novel anti-HSV-2 protein with antiproliferative and peroxidase activities from <italic>Stellaria media</italic>