Fructose 1,6-diphosphatase in normal and diseased human muscle

CLINICA

252

CHIMICA

ACTA

ccrl 4972 Fructose 1,bdiphosphatase in normal and diseased human muscle Various biochemical studies have been made of skeletal muscle from humans with inherited muscular dystrophies. One of the biochemical abnormalities of dystrophic muscle consists in the decrease of overall glycolysis as well as the activities of most of the individual glycolytic enzymesl-5. In contrast, mitochondrial oxidative are not markedly altered, except at the enzymes1 and osidative phosphorylation”*’ very advanced stages, The enzymes involved in glycogenosis are also not much affecteds. It may be hoped that more extensive studies of the biochemical changes in muscle will throw light on the course of events in the evolution of these diseases and perhaps even provide clues col~cerning the initial genetic defect. The present study concerns the activity of fructose ~,6-diphospllatas~ (IDPase) in muscles from normals and from patients with Duchenne muscular dystrophy (D&ID), other forms of dystrophies and a variety of muscular and neuromuscular di+ eases. MATERIALS

AKll METHODS

Cktwaicals Sodium salts of fructose 1,Gdiphosphate (FDP) and fructose 6-phosphate mere purchased from Sigma Chemical Company, St. Louis, MO., U.S.A.

Muscle specimens were biopsies of gastrocnemius,

deltoid or quadriceps

(F6P)

obtained

from II patients with various forms of muscular dystrophy, aged 5-57 years, 5 patients neurogenic atrophies, aged 41-57, I patient with polymyositis, aged 57, and I patient with hypokalemic periodic paralysis, aged 56. The normal samples of deltoid and quadriceps muscle were obtained at surgery on 6 persons, aged 5-63, who showed no evidence of muscle disease. The tissue was homogenized in 9 volumes of cold distilled and deionized water cooling in ice. in an all-glass homogenizer for 2 min with intermittent

A portion of the muscle tissue was routinely examined histologicali~ and rated as “mildly” affected or “severely” affected accordmg to the degree of tissue degeneration. FDPusc assay FDPase (u-fructose I,b-diphosphate I-phosphohydrolase, E.C. 3.1.3.11) was assayed with FDP as substrate as described beforeg. The reaction mixture (0.5 ml) contained qo mmoles glycine buffer, pH o.5, 0.5 mmole MnCl,, 1.2 mmole FDP and 0.1 ml muscle homogenate. The mixture was incubated for IO min at 37”. The reaction was stopped with 0.1 ml of 20% HCIO, and the tubes were cooled in ice and then centrifuged. An aliquot of the centrifugate was anaIyzed far inorganic phosphate by the procedure of Fiske and Subbarowl”. A rough correction for contaminating nonspecific

RRIEF NOTES

253

phosphatases in the homogenates was made by substituting F6P for FDP in the assay and subtracting the value thus obtained from the FDPase assay. In no case was the activity with F6P found to be higher than 20% of that obtained with FDP. Control was kept in which the enzyme was added after incubation. Noncollagm

proteia (NCP)

The NCP was estimated according to a method described beforell. One volume of muscle homogenate was mixed with 9 volumes of 0.05 1%’NaOH and kept for 18 h at room temperature. The protein content of the clear supernate was determined by the method of Lowry & n1.l~. RESULTS

AND DISCUSSION

It can be seen (Table I) that the mean activity of FDPase in normal muscles was not significantly different from that present in the mildly affected muscles from

TABLE:

I

FDPASE

ACTIVITY OFHUMAN

Activity

is expressed

SKELETAL

as nanomoles

MUSCLE

of phosphate No. of cases

Disrase None Muscular dystrophies* Neurogenic atrophies** Palpmyositis Hypokalemic periodic paralysis

6 11 J I

HOMOGENATES

rcleased/mg

NCP/min.

nvgvrc of tissue ~i~~P~z~~at~~~2 .~. ~~ .Mild hIild Severe Severe

FDPasc ~__..___~ 10.-j f 2.rq*** 10.3s & I.O.$ I-‘.85 -& r.rc).+ 5.40

_

I 5.80 _____ ____.-...._ _ _~_.. * Includes these patients: DMD, 3 ; limb girdle dystrophy, 3 ; myotonic dystrophy, 3 ; facioscnpulohumeral dystrophy, I and ocular myopathy, I. ** Includes these patients: Spinal muscular atrophy, z ; unknown neuropathy, 3. * * * Mean & standard

error.

patients with DMD, other dystrophies and neurogenic atrophies. Only in the severely deranged muscles from I patient with pol~myositis and I patient with hypokalemic periodic paralysis was the mean FDPase activity lowered by about 4574, compared to normal. It is well established that FDPase catalyzes an essential step in gluconeogenesis*3. It is present in large amounts in liver and kidney, which are known to be active in gluconeogenesis. The recent demonstration of FDPase activitv in skeletal muscle from a variety of species14,15was somewhat unexpected, since this tissue was not considered to have a significant role in gluconeogenesisl”. However, by comparing the maximum activities of FDPase and phosphofructokinase (PFK) in a variety of muscles in different animals it has been suggestedll that FDPase is present in skeletal muscle to provide cycling between F6P and FDP (catalyzed by PFK and FDPase) when the muscle is at rest. Thus it appears probable that this recycling mechanism may greatly increase the sensitivity of F6P p~~~sphorylation to changes in concentration of AMP. The results presented here show that in contrast to the significantly lowered Clin. Chim. Acta. jP

(1972)

Zj2-2i+

BRIEF NOTES

254 activities of most individual glycolytic enzymes+5, muscle fected in D&ID or in the other dystrophies and neuropathies

FDPase is not at all afexamined.

ACKNOWLEDGEXENTS

This study was supported by U.S. Public Wealth Service Grant by the Muscular Dystrophy Associations of America, Inc.

Received

December

7,

Clirz. Chiru.. AC/U. 38 (1972)

1971 252-254

GM 15759 and