Alkaline and Acid Phosphatase Demonstration in Human Bone and Cartilage: Effects of Fixation Interval and Methacrylate Embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

Alkaline and acid phosphatase demonstration in human bone and cartilage: effects of fixation interval and methacrylate embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Interaction of muscle glycogen phosphorylase b with nicotinic acid, nicotinamide, N-nicotinyl-gamma-aminobutyric acid and nicotinamide coenzymes

The inhibitory action of nicotinic acid, nicotinamide, N-nicotinoyl-gamma-aminobutyric acid, NAD, NADH, NADP, and NADPH on the rabbit skeletal muscle glycogen phosphorylase b has been studied. The inhibition is reversible and positively cooperative (the value of Hill coefficients were determined for the following compounds: nicotinic acid (28 mM; 1.4), nicotinamide (4.4 mM; 1.2), N-nicotinoyl-gamma-aminobutyric acid (9.5 mM; 1.4), NAD (4.4 mM; 1.2), NADH (0.93 mM; 1.2). NADH-binding site of glycogen phosphorylase b subunit was characterized by the sedimentation velocity method. Microscopic dissociation constant was found to be 86 +/- 9 microM (pH 6.8; 20 degrees C). AMP-induced association of glycogen phosphorylase b is hindered by NADH.     

A comparative study of the microsomal S6 phosphatase and phosphorylase phosphatase activities in rat liver.

Rat liver microsomes contain type-1 S6 phosphatase (acting on the serine residues phosphorylated by protein kinase A) and type-1 phosphorylase phosphatase activities. The main aim of this study has been to characterize the microsomal S6 phosphatase activity and to compare its properties with those of the phosphorylase phosphatase activity in the same microsomal preparation. The specific activities of both microsomal S6 phosphatase and phosphorylase phosphatase were 1.6- to 1.7-fold higher in the smooth endoplasmic reticulum than in the rough sarcoplasmic reticulum. Both phosphatase activities were inhibited to a similar extent by MgCl2 (10 mM) and NaF (22 mM), were completely suppressed by glycerophosphate (80 mM) and ZnCl2(10 mM), and were stimulated by MnCl2(1 mM). When analyzed by gel filtration on Sephadex G-100 superfine, both phosphatase activities eluted as broad peaks, stretching from the void volume to 45-60 kDa. The microsomal S6 phosphatase and phosphorylase phosphatase activities also displayed the following distinct characteristics: (a) Mn2+ stimulated the S6 phosphatase activity 2.9-fold more than the phosphorylase phosphatase activity, (b) limited trypsin digestion of microsomal preparations increased the phosphorylase phosphatase activity by 1.5- to 2-fold, but decreased the S6 phosphatase activity by 50%, (c) a synthetic peptide analog of S6 (S6229-239) (200 microM), which did not act as a substrate for the microsomal S6 phosphatase and did not affect its activity, inhibited the microsomal phosphorylase phosphatase activity by about 50%, and (d) the elution profile of the phosphorylase phosphatase activity was markedly broader than that of the S6 phosphatase activity. A series of in vivo studies showed that streptozotocin-diabetes and insulin replacement therapy as well as ip injection of insulin or vanadate, which modified the microsomal S6 phosphatase activity, had no statistically significant effects on the microsomal phosphorylase phosphatase activity. Taken together, these results suggest that the microsomal S6 phosphatase and phosphorylase phosphatase activities are due to two distinct enzyme populations.     

A comparison of several fixative solutions for histochemical studies on human gastric mucosa

The effects of different fixative solutions on the staining of polyanions and Paneth cell granules and on alkaline phosphatase activity were evaluated in surgical specimens of human gastric mucosa with areas of intestinal metaplasia, which were dehydrated and embedded with routine procedures. Alcohol-formol proved to be particularly advisable for studies on the epithelial mucins, buffered formol with cetylpyridinium chloride for the connective tissue polyanions and the fluid of Mota et al. (1956) for the mast cells. In areas of complete intestinal metaplasia, the Paneth cell granules were destroyed by acidic fixative mixtures and 95% ethanol; in the same areas, alkaline phosphatase activity was well demonstrated after fixation with formol, alcohol-formol, or 95% ethanol.     

Evaluation of fixative composition,fixative storage,and fixation duration on the fine structure and volume of root-cell nucleoli

Summary— The effect of various combinations of three fixative compositions (glutaraldehyde buffered in veronal acetate, cacodylate, and piperazine-N, N'-bis[2-ethanesulfonic acid]—PIPES], two fixative storage times (fresh vs 6 weeks), and two fixation durations (3 h vs 9 days) on nucleolar fine structure and nucleolar volume in three root cell-types of oat seedlings (Avena sativa L, cv Seger) were evaluated. All fixatives show overall good preservation of fine structure. Nucleolar components are distinct and well delineated in cells fixed in solutions buffered with either cacodylate or veronal acetate; the components are more condensed when preserved in fixative buffered with PIPES. Nucleolar volume is greatest in cells fixed in the cacodylate fixative, and smallest in those preserved in the PIPES fixative. Among the treatments tested, the PIPES fixative evidently best maintains nucleolar volume. Distracting particulate deposits are abundant on nuclei and nucleoli in cells preserved in the veronal-acetate fixative. Contrary to common assumptions, aging of buffered fixative at room temperature for 6 weeks seems to affect neither the general quality of cellular preservation nor the pH of the fixatives, although nucleolar volume is reduced by such treatment. Long-period fixation (9 days) results in destruction of membrane integrity (mitochondria, plastids, ER), and shrinkage of organelles from the cytoplasm. Nucleolar volume is reduced with prolonged fixation.     

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO₄ fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.Keeping in mind the limitations found, these results open promising perspectives in the study of frozen skeletal muscle samples stored in the tissue banks; this would be especially interesting for rare muscle diseases, where the limited number of biopsies suitable for ultrastructural investigation has so far represented a great restriction in elucidating the cellular mechanisms responsible for the pathological phenotype.Key words: frozen biopsy, electron microscopy, fixation, immunocytochemistry, skeletal muscle.     

An improved method for preparing whole specimens from bovine preimplantation embryos: A technique note

A method was devised to prevent loss of whole embryos during fixation. Specimens were prepared in a chamber saturated with fixative vapors consisting of 3 : 1 (v/v) 96%. ethanol/glacial acetic acid. Good quality specimens were obtained after fixation for at least 24 but not more than 72 h. After staining, specimens could be preserved for 3 to 4 d by storage in the fixation chamber, in 45% aqueous acetic acid vapor. Using the method suggested in this paper prevents loss of early embryos during fixation and allows storage of specimens for longer than usual time while maintaining the quality of the specimen.     

Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme

Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.This study was supported by the Medical Research Service of the Department of Veterans Affairs (TDO), by National Institutes of Health grants No. CA-41267 (LWO), No. HL-39585 and No. HL-44571 (Y-SH), and by the Department of Anesthesiology Research and Development Funds (DBC, HPC).     

Identification of a glycogen synthase phosphatase from yeast Saccharomyces cerevisiae as protein phosphatase 2A.

A glycogen synthase phosphatase was purified from the yeast Saccharomyces cerevisiae. The purified yeast phosphatase displayed one major protein band which coincided with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis. This phosphatase had a molecular mass of about 160,000 Da determined by gel filtration and was comprised of three subunits, termed A, B, and C. The subunit molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 60,000 (A), 53,000 (B), and 37,000 (C), indicating that this yeast glycogen synthase phosphatase is a heterotrimer. On ethanol treatment, the enzyme was dissociated to an active species with a molecular weight of 37,000 estimated by gel filtration. The yeast phosphatase dephosphorylated yeast glycogen synthase, rabbit muscle glycogen phosphorylase, casein, and the alpha subunit of rabbit muscle phosphorylase kinase, was not sensitive to heat-stable protein phosphatase inhibitor 2, and was inhibited 90% by 1 nM okadaic acid. Dephosphorylation of glycogen synthase, phosphorylase, and phosphorylase kinase by this yeast enzyme could be stimulated by histone H1 and polylysines. Divalent cations (Mg2+ and Ca2+) and chelators (EDTA and EGTA) had no effect on dephosphorylation of glycogen synthase or phosphorylase while Mn2+ stimulated enzyme activity by approximately 50%. The specific activity and kinetics for phosphorylase resembled those of mammalian phosphatase 2A. An antibody against a synthetic peptide corresponding to the carboxyl terminus of the catalytic subunit of rabbit skeletal muscle protein phosphatase 2A reacted with subunit C of purified yeast phosphatase on immunoblots, whereas the analogous peptide antibody against phosphatase 1 did not. These data show that this yeast glycogen synthase phosphatase has structural and catalytic similarity to protein phosphatase 2A found in mammalian tissues.     

Time course for cryoprotectant synthesis in the freeze-tolerant chorus frog, Pseudacris triseriata

Increases in liver glycogen phosphorylase activity, along with inhibition of glycogen synthetase and phosphofructokinase-1, are associated with elevated cryoprotectant (glucose) levels during freezing in some freeze-tolerant anurans. In contrast, freeze-tolerant chorus frogs, Pseudacris triseriata, accumulate glucose during freezing but exhibit no increase in phosphorylase activity following 24-h freezing bouts. In the present study, chorus frogs were frozen for 5- and 30-min and 2- and 24-h durations. After freezing, glucose, glycogen, and glycogen phosphorylase and synthetase activities were measured in leg muscle and liver to determine if enzyme activities varied over shorter freezing durations, along with glucose accumulation. Liver and muscle glucose levels rose significantly (5-12-fold) during freezing. Glycogen showed no significant temporal variation in liver, but in muscle, glycogen was significantly elevated after 24 h of freezing relative to 5 and 30 min-frozen treatments. Hepatic phosphorylase a and total phosphorylase activities, as well as the percent of the enzyme in the active form, showed no significant temporal variation following freezing. Muscle phosphorylase a activity and percent active form increased significantly after 24 h of freezing, suggesting some enhancement of enzyme function following freezing in muscle. However, the significance of this enhanced activity is uncertain because of the concurrent increase in muscle glycogen with freezing. Neither glucose 6-phosphate independent (I) nor total glycogen synthetase activities were reduced in liver or muscle during freezing. Thus, chorus frogs displayed typical cryoprotectant accumulation compared with other freeze-tolerant anurans, but freezing did not significantly alter activities of hepatic enzymes associated with glycogen metabolism.     

Enzyme activities of Lyme disease and relapsing fever borreliae

Activities of glycosidases ( n = 8), esterases ( n = 10), arylamidases ( n = 63), acid phosphatase, alkaline phosphatase and phosphoamidase were tested in 47 Borrelia strains. Forty-four were B. burgdorferi strains; 22 of which were isolated from human specimens (skin 13, cerebrospinal fluid six, and one each from blood, heart muscle and synovia), 13 were isolated from various organs of laboratory animals infected via tick bite or with human isolates, and nine from ticks. The remaining three were the relapsing fever strains B. coriaceae , B. hermsii , and B. turicatae. Strains were of low and high passage but the number of subcultures did not influence the enzyme patterns obtained by utilization of chromogenic substrates of constitutive enzymes. Glycosidase activity was absent in almost all strains tested. Esterase activity was high on molecules of chain length 9 carbons. 2-Naphthylamide derivatives were utilized by strains of human, rodent or tick origin in a range of 66.6 to 83.1%. Almost all strains utilized substrates for acid and alkaline phosphatase and phosphoamidase. Chymotrypsin activity was only found in three and two strains from specimens of human and rodent origin, respectively; and γ-glutamyltransferase activity only in three human skin isolates. No strain tested displayed trypsin activity. Overall, the specific activities of constitutive enzymes of the Borrelia strains tested are widely similar. Thus, the enzyme profiles did not discriminate between human, animal and tick isolates, or between human isolates of Borrelia whether cultivated from cerebrospinal fluid or from skin biopsy of Lyme borreliosis patients.     

Inverse relationship of skeletal muscle glycogen from wild-type and genetically modified mice to their phosphorylase a activity.

Leg muscle was biopsied and frozen for storage at -70 degrees C. from 5 wild-type mice, two knocked out acid alpha-glucosidase (GAA) gene mice, and seven glycogen synthase plus glucose muscle transporter transgenic mice. All of the wild-type mice had very little muscle glycogen (3.58 +/- 1.67 micromols glucosyl subunits per g muscle), and 52% or more of its glycogen phosphorylase activity without AMP (69% +/- 17% glycogen phosphorylase a). In contrast the GAA knockout and transgenic mice had glycogen ranging from 63 to 297 micromols glucosyl subunits per g muscle, and very little or no glycogen phosphorylase activity without 1.00 mM AMP (4.8% and less glycogen phosphorylase a). This suggests that there is an inverse relationship between mouse muscle phosphorylase a and the muscle's glycogen content.     

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.     

Influence of two different fixatives on the identification of plasma cells in human rectal mucosa

Plasma cells in sections of bisected human rectal biopsy specimens, fixed in two alternative fixatives, were enumerated after staining by an indirect immunoperoxidase procedure intended to demonstrate immunoglobulin-containing cells. The counts of immunoperoxidase-positive plasma cells were significantly higher after fixation in formol sublimate than after fixation in formol saline. Formol sublimate appears to be a more reliable fixative than formol saline for specimens of rectal mucosa in which quantitation of plasma cells, stained for intracellular immunoglobulin by an immunoperoxidase technique, is intended.     

A cell-free approach to the study of membrane trafficking in frozen rat brains potentially applicable to frozen autopsy material

Summary A cell-free transfer system was used to measure capacity of brain membranes to support membrane renewal. To study transfer in brain, radiolabeled donor microsome fractions were prepared using brain slices from rats or frozen human brain autopsy specimens. Acceptor fractions, prepared from fresh or frozen rat brain or frozen human brain autopsy specimens, were immobilized on nitrocellulose. The complete reconstituted transfer system contained ATP plus ATP-regenerating system (or NADH) as a source of energy and brain cytosol. Slices of frozen brain incorporated acetate into membrane lipids with approximately the same efficiency as fresh brains. This efficiency declined with storage at 4 °C but only slowly. Donor fractions labeled with acetate from frozen slices exhibited specific transfer (37 °C minus 4 °C) of labeled membrane lipids with efficiencies comparable to fresh. The acceptor fraction could be prepared either from fresh or frozen material. Transfer was on the average two-fold stimulated by ATP at 37 °C compared to no ATP. Transfer also was stimulated by NADH. Analysis of linear transfer rates between 0 and 30 min revealed no significant effect of delay time or of time of prolonged storage on transfer efficiency beyond an initial decline of ca. 25% observed within the first two weeks after freezing. A decline of transfer was obtained with brains as the animals aged.     

Characterization of glycogen phosphorylase isoenzymes present in cultured skeletal muscle from patients with McArdle's disease.

Muscle biopsy specimens from patients with McArdle's disease lack glycogen phosphorylase activity. Significant phosphorylase activity was detected in cultured muscle cells from these patients. The phosphorylase isoenzymes in the cells were identified electrophoretically and immunochemically. On polyacrylamide disc gel electrophoresis, two types of isoenzymes were separated in about equal amounts. Both differed the muscle type in migration, kinetic, and immunochemical properties. The first type corresponded to a fetal phosphorylase isoenzyme, and the second was a liver-like type which was completely absorbed with antibody against the rat liver isoenzyme. No adult skeletal muscle isoenzyme was detected.