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The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

ABSTRACT: 1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.

SUBMITTER: Simpson IA

PROVIDER: S-EPMC1164698 | biostudies-other | 1977 May

REPOSITORIES: biostudies-other

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Project description:Thiolated dimeric tetramethylrhodamine (TAMRA) was synthesized in a straightforward procedure utilizing commercially available 5(6)-succinimidyl TAMRA and cystamine hydrochloride. The thiol-containing TAMRA dimer displayed distinct spectral properties in reduced and oxidized forms; covalent dimer formation produced greater effects on the spectral properties than previously reported for noncovalent TAMRA dimers or dimers formed with shorter carbon spacers. The resulting TAMRA disulfide dimer exhibited a hypsochromic shift of 34 nm relative to the reduced monomer species and an isosbestic point at 532 nm between reduced monomeric and oxidized dimeric forms. Molar extinction coefficients of the monomer and dimer relative to moles of TAMRA were similar (6.61 x 10(4) M(-1) cm(-1) and 6.42 x 10(-4) M(-1) cm(-1), respectively). However, fluorescence emission was altered with >93% of dye fluorescence quenched in phosphate buffered saline upon dimer formation. A 520:554 nm absorbance intensity ratio of 2.64 was observed for the oxidized ssTAMRA dimer, almost twice as high as values reported for noncovalent TAMRA dimers. The resulting disulfide dye was easily reduced using both soluble and agarose gel immobilized tris(2-carboxyethyl) phosphine and fresh cell lysate from cultured RAW 264.7 macrophage cells. Absorbance intensity ratios at 554 and 520 nm were used to determine the oxidation half-life of a 1.2 x 10(-5) M solution of reduced TAMRA stored in ambient atmosphere to be approximately 50 h at 22 degrees C. The free thiol dye was further reacted with maleimide-derivatized poly(hydroxypropyl methacrylamide) to yield the dye-labeled polymer conjugate. This dye derivative should prove useful as a dithiol reduction-sensitive fluorescent probe in cellular tracking systems, as well as a thiol-based dye-labeling reagent due to its easy preparation from readily available materials, environmental sensitivity, and simple activation to produce distinct spectral states. The enhanced spectral properties of the covalent TAMRA dimer described here could be useful to prepare more advanced reporter molecules and bioconjugates.

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The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

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