Presence of a Truncated M-Type Subunit and Altered Kinetic Properties of 6-Phosphofructo-l-Kinase Isozymes in the Brain of a Dog Affected by Glycogen Storage Disease Type VII1

Abstract

6-Phosphofructo-1-kinase (PFK) activity in the brain of a dog affected by glycogen storage disease type VII was only 31 % of the PFK activity in the normal dog brain. PFK in the normal dog brain was composed of L-type, M-type and C-type subunits with apparent molecular weights of 78,000, 86,000, and 88,000, respectively, and subunit proportions (L:M:C) of 27:49:24. PFK in the affected dog brain was composed of nearly equal levels of the normal L-type and C-type subunits, but a normal M-type subunit was not detected. Using antidog muscle PFK IgG, immunoblots of gels containing partially purified PFK from the affected dog brain revealed a small amount of immunoreactive protein with an apparent molecular weight of 84,000, suggesting the presence of a truncated M-type subunit. Kinetic studies indicated that the PFK isozymes in the affected dog brain exhibited significantly different kinetic regulatory properties when compared to the PFK isozyme pool in the normal dog brain.

Molecular Basis of Canine Muscle Type Phosphofructokinase Deficiency

“…In contrast to muscle, partially purified extracts of PFK from the erythrocytes and brains of affected dogs showed very small amounts of a lower molecular weight band that was hypothesized to be the truncated M-PFK subunit (14), which has an apparent molecular mass of 84 kDa (32,33). This is 3.3 kDa larger than the predicted molecular weight from the DNA sequence; however, the mobility of proteins in polyacrylamide gels may be affected by factors other than molecular weight, such as glycosylation (34).…”

“…The low residual PFK activity found in muscle (1-4%) could be caused by the mutant M-PFK. Alternatively, there is expression of other PFK isoforms in skeletal muscle (9,14), because in vitro experiments with C2-C12 myogenic cell lines have shown that L-PFK and P-PFK are expressed in myoblasts and at low levels even after myotube formation, indicating that a low expression of these other isoforms in muscle may account for the residual PFK activity (35).…”

“…Minimum essential medium (Eagle's medium-deficient) (Sigma), supplemented with L-leucine, L-lysine, and sodium bicarbonate, but without methionine, was added for 30 min. The medium was removed and deficient medium (above) supplemented with [ 35 S]methionine (Ͼ1000 Ci/mmol, Amersham Corp.) at 5.5 Ci/ml was added for 18 h. The cells were washed with phosphate-buffered saline and either harvested or Dulbecco's modified Eagle's medium was added for periods of up to 24 h. M-PFK was then immunoprecipitated with rabbit anti-dog M-PFK antibody as described and visualized by 7% SDS-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography (12,14).…”

Tissue-dependent loss of phosphofructokinase-M in mice with interrupted activity of the distal promoter: impairment in insulin secretion

“…PFK subunit isoforms were separated using SDS-PAGE and blotted essentially as previously described (54). The primary polyclonal antibodies were rabbit antidog muscle PFK, which recognizes only the M isoform and rabbit anti-rat brain PFK raised against all three isoforms (27).…”

Characterization of phosphofructokinase‐deficient canine erythrocytes

“…A point mutation is reported to occur in the muscle-type gene of deficient dogs, causing a loss of amino acids from the carboxyl MS received 17.1.92 Accepted 15.5.92 terminus of the polypeptide . Studies of brain and erythrocytes from affected (homozygous deficient) dogs indicated that native muscle-type subunits were not present, but small amounts of a structurally unstable truncated muscle-type subunit were found (Mhaskar et al 1991(Mhaskar et al , 1992. As would be expected from the subunit composition of normal tissues, total PFK activity in erythrocytes is about 50% of normal in humans (Vora 1982) and 15-20% of normal in dogs (Giger and Harvey 1987).…”