Dinitrophenyl proteins, preparation

The original evidence for activation by modification of cysteine residues came from studies with 2,4-fluorodinitrobenzene (15). Incubation of the crystalline enzyme preparations with 4 equivalents of FDNB led to a marked increase in activity in the neutral pH range, together with a small decrease in the activity assayed at alkaline pH (Fig. 4). The modified enzyme showed two broad and nearly equal activity maxima one at pH 7.7 and the other at pH 9.0. When dinitrophenylation was carried out at pH 7.5, this change in catalytic properties was associated with the modification of only 2 of the 20 cysteine residues in the protein (16). These 2 highly reactive cysteine residues were found to be completely protected against the action of FDNB by addition of FDP. 

The N-terininal groups in keratin proteins have been estimated using the dinitrophenylation technique of Sanger (1945). Thompson (unpublished observations, 1963) used the method of Steuerle and Hille (1959) and Hille (1960) to determine the N-terminal residues of the three keratose preparations included in Table III. Results similar to those reported in... 

Therefore, in the present study we have measured CPT, activity in mitochondrial, microsomal, peroxisomal and high-speed supernatant fractions prepared from rat liver on a quantitativle basis. We have also quantified the relative expression of proteins that bind dinitrophenyl (DNP)-etomoxiryl-CoA (an oxirane ring-containing inhibitor of hepatic CPT I") in each of these fractions in the intact liver under conditions in which all three activities were optimally inhibited. Our results indicate that the overt CPT activity of microsomes and peroxisomes is associated with a protein that is very similar, if not identical, to mitochondrial CPT I. In microsomes too, the CPT, activity is associated with a protein of Mr 88,(X)0, but in this membrane fraction, there is evidence that the N-terminal domain has different properties from those of mitochondrial and peroxisomal CPT I. 

An essential element in this program is the preparation of pure protein derivatives site specifically labeled by a donor and acceptor and their characterization. Several methods were developed for speci-fic labeling of globular proteins and applied in the preparation of fluorescent derivatives of bovine pancreatic trypsin inhibitor (BPTI) and ribonuclease (RNase). Methoxy-naphthyl derivatives are used as donor chromophores, in particular 2-methoxy-1-naphthyl-methylenyl (MNA) and 2-naphthoxy-acetyl (NAA) were used as donor chromophores (Fig. 3). These two probes are characterized by spectral characteristics suitable for measurements by the present methods (Fig. 4). In particular,they both show monoexponential decay when attached to the protein in the absence of an acceptor under all experimental conditions tested so far. This is a key element in the analysis of fluorescence decay curves of donor-acceptor labeled derivatives, in which all deviations from monoexponential decay are interpreted as distributions of distance. 7-dimethylamino-coumarine-4-acetyl (DA-coum) or 2,4-dinitrophenyl residues were used as acceptors. 

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