PITC derivatives in amino acid analysis

Cohen, Steven A.; Bidlingmeyer, Brian A.; Tarvin, Thomas L.

doi:10.1038/320769a0

Cited by 228 publications

(98 citation statements)

References 12 publications

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“…A sample of the evaporation residue was hydrolyzed (6N HCI, 110°C, 20h, in a sealed tube) and the amino acid composition of the hydrolyzate was analyzed as phenylthiocyanate derivatives by HPLC constructed with a Waters 600 MuItisolvent Delivery System (Waters Association) and Wakopak WS-PTC column (4.0 x 200mm, Wako Pure Chemical Industries). 15,16) …”

Section: Methodsmentioning

confidence: 99%

Caseinphosphopeptides (CPP) in Feces and Contents in Digestive Tract of Rats Fed Casein and CPP Preparations

Kasai

Iwasaki

Tanaka

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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Section: Methodsmentioning

confidence: 99%

Caseinphosphopeptides (CPP) in Feces and Contents in Digestive Tract of Rats Fed Casein and CPP Preparations

Kasai

Iwasaki

Tanaka

et al. 1995

Bioscience, Biotechnology, and Biochemistry

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“…Phenylisothiocyanate derivatization and reverse-phase HPLC 3-methylhistidine formation was determined by phenylisothiocyanate derivatization and reverse-phase HPLC (Waters Pico-tag method; Bidlingmeyer et al, 1984;Cohen et al, 1986) of protein hydrolyzates as described for actin (Raghavan et al, 1989). Peptide fractions, subsequent to the methylation assays described above, were lyophilized, resuspended in water, transferred to hydrolysis tubes along with 5 p1 each of 1 mM standards of I-methylhistidine and 3-methylhistidine.…”

Section: Determination Of 3-methylhistidinementioning

confidence: 99%

The use of alternative substrates in the characterization of actin‐methylating and carnosine‐methylating enzymes

Raghavan

Lindberg

Schutt

1992

European Journal of Biochemistry

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Actin isolated from nearly every eukaryotic species contains approximately 1 mol 3-methylhistidine/ mol protein. His73 in actin has been shown, by protein sequencing, to be the site of methylation. The methylation occurs enzymically and post-translationally. A rabbit skeletal muscle myofibrillary fraction has previously been shown to contain a histidine methyltransferase activity that is actin specific. Detailed study of this enzyme has been hampered by lack of a suitable substrate assay. Naturally occurring actins are poor substrates for the enzyme, presumably due to prexistent methylation at His73. In this study, two potential alternative substrates have been investigated. These are a chicken p-actin expressed in Escherichia coli as a fusion protein with 80 amino acids of an influenza protein, NS1, and a synthetic peptide, Tyr-Pro-Ile-Glu-His-Gly-Ile-Ile-Thr, corresponding to residues 69 -77 of actin. Both substrates were covalently methylated at histidine residues in the presence of S-adenosylmethionine and partially purified enzyme fractions from rabbit muscle. In methylation experiments employing the fusion actin in the form of inclusion bodies, 3-methylhistidine is the major product, as is the case when soluble muscle or non-muscle actin is used. However, for the synthetic peptide, the methylated product primarily contained 1-methylhistidine and only a small amount of the isomeric 3-methylhistidine. Further investigations revealed that the peptide was recognized by carnosine N-methyltransferase, another histidine methyltransferase found in muscle tissue. Carnosine N-methyltransferase appears to copurify with the actin-methylating enzyme in preliminary fractionation experiments. Separation of the two methyltransferase activities is described.

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“…Unless otherwise stated, proteins subjected to polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) were analyzed in 12.5% gels as described by Laemmli (19) (15, Amino acid analysis. Phenylisothiocyanate derivatives from acid hydrolysates of purified inclusions were prepared as described by Cohen et al (9). Samples were analyzed by using a PICO-TAG amino acid analysis system (Waters Associates).…”

Section: Methodsmentioning

confidence: 99%

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus

Couche¹,

Gregson²

1987

J Bacteriol

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The entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus produces two types of intracellular inclusion bodies during in vitro culture. Large cigar-shaped inclusions (designated type 1) and smaller ovoid inclusions (designated type 2) were purified from cell lysates, using differential centrifugation in discontinuous glycerol gradients and isopycnic density gradient centrifugation in sodium diatrizoate. The inclusions, composed almost exclusively of protein, are readily soluble at high and low pH values and in the presence of cation chelators such as EDTA, anionic detergents (sodium dodecyl sulfate), or protein denaturants (urea, NaBr). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inclusions revealed a single 26-kilodalton protein (IP-1) in type 1 inclusions and a 22-kilodalton protein (IP-2) in type 2 inclusions. Analysis of these proteins by isoelectric focusing in the presence of 8 M urea showed that IP-1 is acidic and IP-2 is neutral. Furthermore, each protein occurred in multiple forms differing slightly in isoelectric point. Other variations in peptides released by trypsin digestion, immunological properties, and amino acid composition revealed significant structural differences between IP-1 and IP-2. Kinetic studies using light microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting procedures showed that inclusion protein synthesis occurs only during the second half of exponential culture growth. Synthesis of inclusion proteins and their aggregation to form inclusions occurred concurrently. Possible functions for these abundant proteins are discussed.Xenorhabdus species are entomopathogenic bacteria symbiotically associated with insect parasitic nematodes of the families Heterorhabditidae and Steinernematidae (1,3,30). The bacterial symbionts are asporogenous gram-negative rods, which are carried monoxenically in the intestine of nonfeeding infective-stage nematodes. On penetration of an insect host, the bacteria are released into the hemocoel. Colonization by the bacteria kills the host and establishes a suitable environment for reproduction of the nematodes by providing nutrients and inhibiting the growth of other microorganisms (1,24).When cultured in vitro, Xenorhabdus bacteria occur in two forms designated primary and secondary, which can be distinguished according to colony morphology and pigmentation on various bacteriological media, or on the basis of antibiotic production (1, 2). Both forms are pathogenic to insects, but only the primary form is isolated from infective nematodes (1). Xenorhabdus spp. will grow in a wide variety of artificial media which are rendered suitable for nematode reproduction, thus providing the basis for economical mass production of nematodes. Our nematophilus, in contrast to X. luminescens in which both primary and secondary forms produced inclusions (10). Intracellular inclusion bodies have also been described in several ultrastructural studies of Xenorhabdus spp. (4,7,15,18).In this repor...

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PITC derivatives in amino acid analysis

Abstract: Refined methods for separating PTC-amino acids on reverse phase columns may pose a challenge to traditional ion exchange techniques.

Cited by 228 publications

References 12 publications

Caseinphosphopeptides (CPP) in Feces and Contents in Digestive Tract of Rats Fed Casein and CPP Preparations

Caseinphosphopeptides (CPP) in Feces and Contents in Digestive Tract of Rats Fed Casein and CPP Preparations

The use of alternative substrates in the characterization of actin‐methylating and carnosine‐methylating enzymes

Protein inclusions produced by the entomopathogenic bacterium Xenorhabdus nematophilus subsp. nematophilus

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