Phosphofructokinase: Studies on the subunit structure

The Subunits of Rabbit-Muscle Phosphofructokinase. A Search for Sequence Repetition

“…The criteria of chemical purity were that the enzyme was electrophoretically homogeneous when examined by polyacrylamide gel-electrophoresis in 0.1 "/, sodium dodecylsulphate as well as in 6 M urea at three different pH values. The preparations used were also devoid of N-terminal residues, in agreement with previous work [ 3 ] , and showing that they did not contain internal cleavages caused by partial proteolysis during isolation as has been shown to occur in the case of yeast phosphofructokinase [31].…”

“…This independent estimate for the cysteine content of rabbit muscle phosphofructokinase is in excellent agreement with values determined by amino acid analysis in this (cf. Table 1) and in previous [3] studies. Values obtained for lysine (43) and arginine (53) are also in good agreement with previous results and indicate a total of approximately 100 trypsinsensitive bonds per polypeptide chain.…”

“…Peptides revealed by autoradiography and selective staining were thus not representative of the entire protein chain and this procedure would therefore have given a gross underestimate of the total number of tryptic peptides in the digest. In this connection it may be recalled that previous attemps to prepare peptide maps of the enzyme by methods involving the use of electrophoresis at pH 6.5 were reported [3,5] to have given rise to approximately half the expected number of tryptic peptides. Moreover it was these findings that led Brennan et ul.…”

“…In earlier studies of the subunit structure of rabbit muscle phosphofructokinase, Paetkau et al [3] noted that the number (40-50) of peptides found in a tryptic digest of the enzyme was approximately half the number to be expected from a polypeptide chain with a molecular weight of 85000 and containing about 100 potential sites for tryptic cleavage. Subsequently Coffee et al [4] found only 8-9 unique radioactively labelled cysteine-containing peptides in a tryptic digest of S-carb~xy['~C]methylated rabbit muscle enzyme, half the number (16 -17) to be expected from the amino acid composition were the subunit polypeptide chain to be of unique sequence over its entire length.…”

The Self‐Association of Rabbit‐Muscle Phosphofructokinase

“…At the present stage of the analysis, bearing in mind the errors involved in the equilibrium experiments, we suggest that the self-association of phosphofructokinase may be described by the following equations : The discrepancy between their results and that obtained here is not readily explicable. It is possible that phosphofructokinase is a difficult enzyme to denature completely and that our conditions were not as drastic as those of Paetkau et al [19], although on the face of it they appear identical. The previous history of the enzyme preparation may also affect the ease with which it denatures.…”