Wheat-embryo ribonucleates. XV. Characterization of a fraction of RNA subject to conspicuous terminal labeling during early germination of wheat embryos

1984 ◽  
Vol 62 (6) ◽  
pp. 321-328 ◽  
Author(s):  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Amino Acids ◽  
Gel Filtration ◽  
Isotopic Labeling ◽  
Developmentally Regulated ◽  
Wheat Embryo ◽  
Physiological Importance ◽  
5.8S Rrna ◽  
Early Germination ◽  
Wheat Embryos

Various techniques have been used to characterize the rapidly migrating electrophoretic components (RMEC) in an RNA fraction which is subject to conspicuous terminal labeling (mostly adenosine) when wheat embryos are pulse labeled with 3H-labeled nucleosides during early germination. Detection of terminally labeled RMEC RNA is most favoured during early germination when labeling of 3′-hydroxyl termini in preexisting RNA, catalyzed by RNA nucleotidyltransferases, is greatest in relation to nonterminal labeling of nascent RNA, catalyzed by RNA polymerases. The RMEC RNA is tenaciously associated with a fraction of high-molecular-weight RNA and it can be subdivided and classified into two fractions, RMEC-1 and RMEC-2, by electrophoresis in polyacrylamide, gel filtration through Sephadex, or assay of amino acid acceptance. The RMEC-1 RNA and bulk wheat-embryo tRNA have similar capacities to accept a wide variety of amino acids, but RMEC-2 RNA does not accept any of the amino acids tested. RMEC-2 RNA is broadly heterodisperse and one of its component polynucleotides has been identified by sequence analysis as a tridecanucleotide fragment from the 3′-hydroxyl end of 5.8S rRNA. This study was undertaken as part of a broader program in which isotopic labeling and analysis of nucleates and proteins in germinating wheat embryos have been used to detect signal events and to evaluate their physiological significance. It is concluded that conspicuous terminal labeling of RMEC RNA during early germination of wheat embryos is unlikely to be of physiological importance. However, isotopic labeling and analysis of the nucleates and proteins in germinating embryos has uncovered other signal events which appear to be of physiological importance. Discovery of the existence of a number of developmentally regulated proteins and the way in which discovery of these proteins is expected to direct the course of future investigations in the laboratory are subjects of a brief discussion.

Wheat-Embryo Ribonucleates. IV. Factors That Influence the Formation and Stability of a Complex Between 5S rRNA and 18S rRNA

1975 ◽  
Vol 53 (3) ◽  
pp. 320-327 ◽  
Author(s):  
A. A. Azad ◽  
B. G. Lane
Keyword(s):  
18S Rrna ◽  
5S Rrna ◽  
23S Rrna ◽  
Wheat Embryo ◽  
Physiological Importance ◽  
5.8S Rrna ◽  
Wheat Embryos ◽  
The Subject ◽  
26S Rrna ◽  
Source Materials

Under the conditions used in this study, wheat-embryo 5S rRNA complexes with its homologous 18S rRNA from wheat embryos and with heterologous 18S rRNA from other eukaryotic source materials such as yeast, L cells, and HeLa cells, but it does not complex with heterologous 16S rRNA from a prokaryote such as Escherichia coli or with homologous or heterologous 26S(23S) rRNA of either eukaryotic or prokaryotic origin.If a solution of wheat-embryo rRNA is simply made 0.3 M with respect to NaCl and then heated at 60 °C for 3 min before quick cooling to room temperature (ca. 20 °C), there is both preferential and efficient complex formation between 5S and 18S rRNA and between 5.8S and 26S rRNA.The laboratory-prepared' complex between wheat-embryo 5S rRNA and its homologous 18S rRNA is more thermostable in 0.1 M NaCl solution than is the 'natural' complex between wheat-embryo 5.8S rRNA and its homologous 26S rRNA, and both complexes 'melt' over a narrow range of temperature.The possible physicochemical and physiological importance of both homologous and heterologous rRNA complexes is the subject of a brief discussion.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids

1980 ◽  
Vol 185 (2) ◽  
pp. 373-381 ◽  
Author(s):  
N D Light ◽  
A J Bailey
Keyword(s):  
Amino Acids ◽  
Type I Collagen ◽  
Gel Filtration ◽  
Amino Acid Content ◽  
Exchange Chromatography ◽  
Type I ◽  
Polymeric Form ◽  
Cross Links ◽  
Helical Region

A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 × 10(6)-5 × 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.

scholarly journals Purification and characterization of two wheat-embryo protein phosphatases

1988 ◽  
Vol 251 (2) ◽  
pp. 357-363 ◽  
Author(s):  
G M Polya ◽  
M Haritou
Keyword(s):  
Protein Phosphatase ◽  
Wheat Germ ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Dependent Protein Kinase ◽  
Wheat Embryo ◽  
Dependent Protein ◽  
Enzyme I

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.

scholarly journals Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum

1981 ◽  
Vol 199 (1) ◽  
pp. 9-15 ◽  
Author(s):  
M Janusz ◽  
K Starościk ◽  
M Zimecki ◽  
Z Wieczorek ◽  
J Lisowski
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Room Temperature ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Guanidinium Chloride ◽  
Terminal Amino Acid ◽  
Polyproline Ii ◽  
Terminal Amino

A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

CHARACTERIZATION OF AMINO ACID AND CARBOHYDRATE COMPONENTS IN FULVIC ACID

1976 ◽  
Vol 56 (3) ◽  
pp. 159-166 ◽  
Author(s):  
G. GUIDI ◽  
G. PETRUZZELLI ◽  
P. SEQUI
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Fulvic Acid ◽  
Weight Distribution ◽  
Gel Filtration ◽  
Percentage Content ◽  
Acidic Amino Acids ◽  
Carbohydrate Components

The distribution of individual amino acids and monosaccharides in fulvic acid and its fractions separated by polyamide chromatography was investigated in five different Italian soils. Although little differences were generally found in the two polyamide fractions (FI and FII), the highest percentage content of acidic amino acids and the lowest percentage content of neutral amino acids have been found in the second one (FII); monosaccharides composition was more irregular, but generally FII contained more pentoses. Both chromatographic fractions (FI and FII) have been chromatographed on Sephadex G-25. The composition in carbohydrate and amino acid components of the further different fractions resolved by gel filtration showed great differences depending on the molecular weight distribution.

scholarly journals Isolation and characterization of a hydroxyproline-containing protein from soluble extracts of the leaves of sandal (Santalum album L.)

1974 ◽  
Vol 141 (1) ◽  
pp. 147-153 ◽  
Author(s):  
Uliyar V. Mani ◽  
Amurtur N. Radhakrishnan
Keyword(s):  
Amino Acids ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Partial Hydrolysis ◽  
Santalum Album ◽  
Isolation And Characterization ◽  
Approximate Molecular Weight ◽  
Santalum Album L

1. A hydroxyproline-containing protein was isolated from the soluble fraction of sandal leaves (Santalum album L.) and the purified protein was homogeneous by disc electrophoresis. 2. It is a glycoprotein containing 16% carbohydrate, the components of which were mainly arabinose, with only small amounts (about 5%) of galactose. The principal amino acids were glutamic acid, aspartic acid, glycine, alanine, arginine, lysine, proline and hydroxyproline, which together comprised 60% of the total. The number of acidic amino acids exceeds the number of basic amino acids. By Sephadex gel filtration, the approximate molecular weight was found to be about 63000. The ratio of residues of hydroxyproline to those of arabinose was 1:2. 3. The native protein is resistant to the action of several proteolytic enzymes. After partial hydrolysis with 0.1m-HCl, the protein became susceptible to attack by Pronase but remained resistant to collagenase.

Purification and characterization of two xylanases from Chaetomium thermophile var. coprophile

1989 ◽  
Vol 35 (9) ◽  
pp. 836-842 ◽  
Author(s):  
Ramesh K. Ganju ◽  
Paul J. Vithayathil ◽  
S. K. Murthy
Keyword(s):  
Amino Acids ◽  
Enzyme Purification ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Larch Wood ◽  
Hydrolysis Products ◽  
Xylanase Enzyme ◽  
Temperature Optima

Two xylanases (I and II) out of several extracellular xylanases produced by the thermophilic fungus Chaetomium thermophile var. coprophile were purified to homogeneity by a combination of ion exchange and gel filtration chromatographic procedures. They had molecular weights of 26 000 (xylanase I) and 7000 (xylanase II). The temperature optima for xylanase I and II were 70 and 60 °C, and they were optimally active at pH 4.8–6.4 and 5.4–6.0, respectively. Xylanase I was found to be comparatively more stable than xylanase II at higher temperatures. Amino acid composition indicated that xylanase I contained high amounts of glycine, threonine, and low amounts of histidine and sulphur-containing amino acids. Each enzyme released different hydrolysis products from larch wood xylan. Xylanase I produced mainly xylobiose and xylotriose whereas xylanase II produced mainly xylobiose.Key words: Xylanase, enzyme purification, characterization, Chaetomium thermophile.

Wheat embryo ribonucleates. XII. Formal characterization of terminal and penultimate nucleoside residues at the 5′-ends of 'capped' RNA from imbibing wheat embryos

1978 ◽  
Vol 56 (7) ◽  
pp. 729-733 ◽  
Author(s):  
M. H. Haffner ◽  
M. B. Chin ◽  
B. G. Lane
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Animal Cells ◽  
Higher Plant ◽  
Wheat Embryo ◽  
Wheat Embryos

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains [methyl-3H]methionine.(2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of 'capped' oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2.(3) By way of contrast with the 'capped' RNA of animal cells, 5′-terminal 'cap' structures (m7Gppp- and m32,2,7Gppp-) in the 'capped' RNA from the higher plant organism are not bonded to penultimate O2′-methylnucleoside constituents.(4) In an allied study, it has been found that recovery of poly(A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly(A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris(hydroxymethyl)aminomethane buffered (pH 9.0) phenolic emulsions.

scholarly journals The use of acid alumina and Sephadex LH-20 for the separation and characterization of ethanol-soluble peptides produced by Bacillus brevis

1972 ◽  
Vol 127 (3) ◽  
pp. 489-502 ◽  
Author(s):  
I. M. Bartley ◽  
B. Hodgson ◽  
J. S. Walker ◽  
G. Holme
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Bacillus Brevis ◽  
Water Solvent ◽  
Cell Extracts ◽  
Layer Chromatography

Cell extracts from Bacillus brevis (A.T.C.C. 10068), grown with various media, incorporated certain 14C-labelled amino acids that are normally components of tyrothricin into material that was extracted by ethanol from the precipitate formed by adding acid. When this material was separated by paper and silica-gel thin-layer chromatography and paper electrophoresis 14C was located in those regions that also contained gramicidin and tyrocidine. From a study of the properties of the system responsible for the incorporation it was deduced that non-tyrothricin materials were present. It was shown that the methods normally used to characterize tyrothricin do not adequately distinguish between tyrothricin and non-tyrothricin materials. However, a method for separating these materials was devised. This involved elution with ethanol from columns of acid alumina followed by gel filtration on Sephadex LH-20 with dimethylformamide–water solvent. The behaviour of gramicidin and tyrocidine on the Sephadex LH-20 column was examined, and it was concluded that the separation was not caused simply by gel filtration of unassociated molecules. Also, tyrocidine molecules with different amino acid compositions seemed to have different affinities for the Sephadex LH-20 column.

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