Rat Muscle Extract Research Articles

Enhanced fusion of myoblasts with myofibers for efficient gene delivery induced by a partially purified protein fraction from rat muscle extract

The biggest challenge to gene therapy is how to efficiently deliver the desired therapeutic gene into a sufficient number of recipient cells to achieve significant clinical efficacy. Here, we identified a partially purified extract from rat muscle probably containing myoblast specific fusion factor(s) (MSF), which significantly enhanced fusion of donor myoblast with host muscle fibers. Once incorporated, the introduced genetic construct could instruct the machinery of the hybrid cells to express the desired protein(s). Rat satellite cells containing a plasmid carrying a marker bone morphogenetic protein-4 (BMP-4) coding sequence were used as foreign gene delivery vehicle. BrdU labeling of the MSF-pretreated satellite cells allowed tracing the fate of the genetically modified satellite cells in the host muscles. Immunohistochemistry using anti-BMP-4 antibody demonstrated the translation of the introduced gene construct. It was demonstrated that in the presence of MSF, numerous BrdU positive nuclei and the expression of BMP-4 polypeptides could be observed in host hybrid fibers, while in the control group using rat serum to replace MSF containing fraction, only a few BrdU positive signals were detected. The expression of osteocalcin and the elevated alkaline phosphatase activity detected in the hybrid fibers indicated the proper folding, secretion and, post-translational modification of the expressed foreign protein. This strategy of enhanced myoblast-mediated gene transfer would break the major barrier in current practice of normal or engineered myoblast transplantation in the management of genetic muscle diseases or systemic genetic disorders.

Receptors to Steroid Hormones and Aromatase Are Expressed by Cultured Motoneurons but Not by Glial Cells Derived from Rat Embryo Spinal Cord

The aim of this study was to examine the expression of aromatase and receptors to steroid hormones in cultured motoneurons (MNs). We first developed an original method for obtaining rat MN cultures. Dissociated E15 rat spinal cords were purified using metrizamide and bovine serum albumin density gradients, and cells were then seeded on the culture substratum. We optimized the culture parameters and found that simple addition of rat muscle extract (ME) and conditioned culture medium (CM) from glial cell lines (GCL) derived from spinal cord were sufficient to obtain almost pure MN cultures. MNs were characterized by the presence of specific MN markers and electrophysiology. MNs could be kept alive for 2 weeks. We demonstrate that ME and CM are essential for MN development and survival respectively. Immunocytochemistry and aromatase activity assay indicated the presence of androgen and estrogen receptors as well as aromatase in MNs but not in GCL. This is the first report demonstrating the presence of both female and male sex hormone receptors and a key enzyme in steroid hormone metabolism in MNs and its absence in GCL, at least in our culture conditions. This in vitro model appears to be valuable for elucidating the impact of the sex hormone circuit in neuronal maturation. The relevance of this model for the comprehension of neurodegenerative diseases is discussed.

The activation of glycolysis performed by the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the model system

Influence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) on glycolysis was investigated. The addition of GAPN—which oxidizes glyceraldehyde-3-phosphate directly to the 3-phosphoglyceric acid—led to the strong increase in the rate of lactate accumulation in the rat muscle extract with low ADP content. The lactate accumulation was also observed in the presence of GAPN in rat muscle extract, which contained only ATP and no ADP. This can be the evidence of the “futile cycle” stimulated by GAPN. Here ADP can be regenerated from ATP by the phosphoglycerate kinase reaction. The high resistance of GAPN from Streptococcus mutans towards inactivation by natural oxidant—H 2O 2 was showed. This feature distinguishes GAPN from phosphorylating glyceraldehyde-3-phosphate dehydrogenase, which is very sensitive to modification by hydrogen peroxide. A possible role of the oxidants and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the regulation of glycolysis is discussed.

Muscle Develops a Specific Form of Small Heat Shock Protein Complex Composed of MKBP/HSPB2 and HSPB3 during Myogenic Differentiation

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.

Insulin Receptor Substrate Proteins Create a Link between the Tyrosine Phosphorylation Cascade and the Ca2+-ATPases in Muscle and Heart

Following phosphorylation by the insulin receptor kinase, the insulin receptor substrates (IRS)-1 and IRS-2 bind to and activate several Src homology 2 (SH2) domain proteins. To identify novel proteins that interact with IRS proteins in muscle, a human skeletal muscle cDNA expression library was created in the lambdaEXlox system and probed with baculovirus-produced and tyrosine-phosphorylated human IRS-1. One clone of the 10 clones which was positive through three rounds of screening represented the C terminus of the human homologue of the adult fast twitch skeletal muscle Ca2+-ATPase (SERCA1) including the cytoplasmic tail and part of transmembrane region 10. Western blot analysis of extracts of rat muscle demonstrated co-immunoprecipitation of both IRS-1 and IRS-2 with the skeletal muscle Ca2+-ATPase (SERCA1) and the cardiac muscle isoform (SERCA2). In both cases, injection of insulin stimulated a 2- to 6-fold increase in association of which was maximal within 5 min. In primary cultures of aortic smooth muscle cells and C2C12 cells, the insulin-stimulated interaction between IRS proteins and SERCA1 and -2 was dose-dependent with a maximum induction at 100 nM insulin. This interaction was confirmed in a "pull down" experiment using a glutathione S-transferase fusion protein containing the C terminus of the human SERCA isoform and phosphorylated IRS-1 in vitro and could be blocked by a FLVRES-like domain peptide present in the human SERCA sequence. Affinity chromatography of phosphopeptide libraries using the glutathione S-transferase fusion protein of the C terminus of SERCA1 indicated a consensus sequence for binding of XpYGSS; this is identical to potential tyrosine phosphorylation sites at position 431 of human IRS-1 and at position 500 of human IRS-2. In streptozotocin diabetic rats the interaction between IRS proteins and SERCA1 in skeletal muscle and SERCA2 in cardiac muscle was significantly reduced. Taken together, these results indicate that the IRS proteins bind to the Ca2+-ATPase of the sarcoplasmic reticulum in an insulin-regulated fashion, thus creating a potential link between the tyrosine phosphorylation cascade and effects of insulin on calcium.

Characterization of a Human T Cell Line Reactive to a 52 kDa Islet Protein

A 52 kDa islet protein has recently been identified as the target of autoantibodies in the NOD mouse model of IDDM and humans with IDDM. However, the presence of T cell immunity against the 52 kDa islet protein in IDDM has not been reported. We report the establishment and characterization of a T cell line (19KW) that reacts to purified 52 kDa islet protein (purified p52) from a subject with IDDM. The purified p52 induced a proliferative response as measured by thymidine incorporation in the 19KW T cell line with a stimulating index of up to 48. The proliferative responses were greater with increasing doses of purified p52 (0.1, 0.5, 2.0, and 6.0 μg/well). No reactivity was found to a liver fraction purified in the same manner as 52 kDa protein, BSA, ovalbumin, extracts of rat muscle, fibroblast, adrenal, or pituitary tissue and to a rat exocrine cell tumor. Irradiated PBMC were required as antigen presenting cells (APC) for 19KW reactivity to the purified p52. The addition of anti-HLA DR or anti-HLA DQ antibodies significantly decreased the islet antigen-induced proliferative response. The addition of antibodies to HLA DP and class I MHC had no effect. Flow cytometric analysis revealed that the majority of T cells expressed CD4 and CD45RO molecules. T cell receptors Vβ6 and Vβ5.1 were found on 30 and 14% of the CD3+(T cells) 19KW cells, respectively. In conclusion, a purified p52-reactive human T cell line predominantly consisting of TCR Vβ6+and Vβ5.1+cells has been established from a subject with IDDM. Reactivity to the purified p52 is antigen dose-dependent, tissue specific, requires irradiated PBMC as antigen presenting cells, and is HLA DR- and HLA DQ-restricted. T cell lines specifically reactive to p52 may be useful for investigating further the role of this antigen in the pathogenesis of IDDM.

Proteins in tissue extracts which bind insulin-like growth factor binding protein-3 (IGFBP-3).

Membrane associated IGFBP-3 is now known to play a role in the modulation of IGF at the cellular level, but mechanisms involved in cell membrane binding are far from certain. In this study we report the identification and initial structural characterisation of proteins in a range of sheep and rat tissues which specifically bind recombinant human non-glycosylated IGFBP-3. Tissues were homogenised in Tris HCl (0.1 M, pH 7.4), containing proteolytic enzyme inhibitors and the residues re-extracted in buffer containing SDS and Triton X-100 (both 1% w/v) prior to analysis. These were subjected to SDS-PAGE, electro-blotted onto nitrocellulose and subjected to ligand blot analysis (LBA) using radioiodinated IGFBP-3 as ligand. LBA revealed a major band of binding activity migrating at 60 kDa in extracts of rat muscle while sheep muscle contained forms of 52 and 40 kDa as the principal species and ovine pancreatic extracts an abundance of the 40 kDa variant alone. Distribution of the binding activity appears tissue specific. Apart from skeletal muscle, pancreas and a small amount of the 52 kDa form in pituitary, analysis revealed no evidence of IGFBP-3 binding in a range of other sheep tissues including liver, kidney, spleen, intestine, adrenal, brain, mammary, uterus, ovary and plasma. The binding activity is TCA precipitable, trypsin digestible, dose responsive and relatively specific for IGFBP-3 since the related proteins recombinant human IGFBP-2 and purified caprine IGFBP-4 failed to bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosinatable and non-tyrosinatable tubulin subpopulations in rat muscle in comparison with those in brain.

Using immunobinding and enzymatic assays we determined in rat muscle extracts the proportion of tyrosinatable tubulin, that is, tubulin that participates in the tyrosination/detyrosination cycle. We found that in muscle, in contrast with nervous tissue, practically all tubulin molecules are tyrosinatable. In the case of rat brain the non-tyrosinatable tubulin pool accounts for about 50% of the tubulin. In addition, isolectrofocusing of 14C-tyrosinated tubulin from brain and muscle extracts revealed a different composition in tyrosinatable tubulin isotypes. One of the isotypes, which in muscle accounts for 86% of the 14C-tyrosinated tubulin species, was detyrosinated by the action of tubulin carboxypeptidase faster than the rest of the 14C-tyrosinated tubulin isotypes taken in whole. In the case of brain extract, that isotype accounts for only 16% of the labeled tubulin.

Myosin heavy‐chain‐based isomyosins in developing, adult fast‐twitch and slow‐twitch muscles

A modified method of electrophoresis under nondenaturing conditions made it possible to separate rat muscle extracts of defined myosin heavy chain (HC) and light chain (LC) composition into subsets of developmental, fast and slow myosin heavy-chain-based isomyosins. The fastest migrating isomyosins were the neonatal isomyosins (nM1, nM2, nM3), followed by the slightly slower migrating embryonic isomyosins (eM1, eM2, eM3, eM4). Of the nine adult fast isomyosins, the HCIIb-based isomyosins (FM1b, FM2b, FM3b) were the fastest migrating. These were followed by the HCIId-based isomyosins (FM1d, FM2d, FM3d). The HCIIa-based isomyosins (FM1a, FM2a, FM3a) were the slowest. Our results suggest that FM3a is identical with the so-called intermediate isomyosin (IM) described in the literature. The slow myosin heavy-chain-based isomyosins (SM1, SM2, SM3) migrated far behind the fast isomyosins. Whereas the gross electrophoretic mobilities of each of these isomyosin triplets is determined by the specific heavy chain complement, the different mobilities of the bands within each triplet result from different alkali light chain combinations. Thus, the fastest triplet bands of the neonatal (nM1) and adult fast isomyosins (FM1b, FM1d, FM1a) represent the LC3f homodimers, the slowest (nM3, FM3b, FM3d, FM3a) the LC1f homodimers, and the intermediate bands (nM2, FM2b, FM2d, FM2a) the LC1f/LC3f heterodimers. Different proportions of the adult fast isomyosin triplet bands indicate that the affinity for LC3f decreases in the order HCIIb, HCIId, HCIIa. The three slow isomyosins represent LC1sa (SM1) and LC1sb (SM3) homodimers and a LC1sa/LC1sb heterodimer (SM2). Circumstantial evidence suggests an inverse order in rabbit muscle where SM1 and SM3 most likely represent LC1sb and LC1sa homodimers, respectively.

Method for isolation of MMP-7-ase isoenzymes from rat muscle tissue extract.

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HPLC columns. The procedures involved are extraction, ultracentrifugation, chromatographies and enzyme assays and require less than five hours.

Distribution of metabolic fluxes towards glycerol phosphate and l-lactate from fructose 1,6-biphosphate in vitro: Effect of glycerol phosphate dehydrogenase

1. 1. A metabolic system in vitro, which converts fructose 1,6-biphosphate into the two alternative products, lactate and glycerol phosphate, was designed to study the distribution of metabolic fluxes and, specifically, the control of glycerol phosphate production rate in rat muscle extract. 2. 2. Experiments were carried out at several protein concentrations by dilution of rat muscle extract, showing non-linear behaviours of flux versus protein concentration. These were hyperbolic for glycerol phosphate and logarithmic for l-lactate. 3. 3. The influence of the flux towards any alternate product on the rate giving the other was studied by stimulation of each. Results obtained show that in this system, flux towards glycerol phosphate is not affected by lactate production and the same occurs for the contrary case. 4. 4. Glycerol phosphate dehydrogenase seems to be the only enzyme in this system whose activity controls the flux towards glycerol phosphate.

Demonstration of anti-muscle immunity in murine trichinellosis

Swiss mice infected with Trichinella spiralis (I mice), or immunized with straight Freund's complete adjuvant (A mice) or with rat muscle extract (RME) in Freund's complete adjuvant (MA mice) were tested for skin reactivity against RME and compared with uninfected, unimmunized, tested controls (C mice). C, A and I mice infected for 32 days did not show any reaction; MA mice exhibited significant enlargement of the foot-pads at 24 h of the skin inoculation when tested 7 days after immunization and at 0.5, 5, 24 and 48 h of inoculation when tested 18 days after immunization. I mice infected for 3 months produced skin reactions at 0.5, 5 and 48 h of the inoculation. RME is antigenic in mice when injected with adjuvant and 3 month old T. spiralis infection elicits immediate, intermediate and delayed skin reactivities to heterologous muscle components.

The control of glucose 1,6-bisphosphatase by Ca2+ and calmodulin.

1. The control of glucose 1,6-bisphosphatase activity, the enzyme that degrades glucose 1,6-bisphosphate, a metabolite that regulates hexose phosphate metabolism, has been examined in a rat muscle extract. 2. The enzyme has been found to be activated by physiological Ca2+ concentrations. Ca2+ can be replaced by Sr2+ and Mn2+, but the effects are not so pronounced. 3. The Ca2+ effect is inhibited by EGTA. Trifluoperazine also inhibits enzyme activity. This effect can be reversed by adding exogenous calmodulin.

Zinc and carbonic anhydrase III distribution in mammalian muscle

Zinc and carbonic anhydrase III measurement in human and rat muscle extracts indicate that: 1. 1. About one fifth of zinc in human soleus is associated with carbonic anhydrase III isozyme, and even higher levels of zinc and carbonic anhydrase III are found in rat soleus, where about one half of the zinc is in carbonic anhydrase III. Other muscle was also analysed in a similar way, (see text). Heart is notable in containing lower levels of zinc but negligible carbonic anhydrase III. 2. 2. Treatment of muscle with water or phosphate solutions showed that all the carbonic anhydrase III was water extractable, whereas significant zinc remained bound, but was partially extractable by phosphate solutions. 3. 3. Dialysis of muscle extracts showed that whilst some zinc was dialysable, there was no significant contribution from the carbonic anhydrase III in the dialysed extract. EDTA enhanced the release of dialysable zinc from muscle extract. These findings are discussed in relation to muscle disease.

Comparative studies on AMP-deaminase—V. Change of enzyme activity during storage

1. 1. The specific activities of homogenous AMP-deaminases isolated from rat, rabbit, hen, frog and perch skeletal muscle increased during storage at 4°C up to 2 weeks. A longer storage caused gradual decrease of activity. 2. 2. No change of specific activity was observed during storage of the enzymes from human and pikeperch skeletal muscle and from frog liver. 3. 3. The absorbancy ratio A 280/A 260 was in the range 1–2 for each enzyme studied, being stable during storage of a given enzyme preparation. 4. 4. Limited proteolysis with trypsin resulted in a significant increase of activity of AMP-deaminase in crude extracts of rat muscle and hen white as well as red muscles. 5. 5. Upon trypsin treatment the specific activity of AMP-deaminase purified from rat muscle increased while negligible activity increase with the enzymes purified from hen white and red muscles was observed.

Lymphocyte responsiveness to acetylcholine receptor in rats with experimental autoimmune myasthenia gravis.

We examined the time course of lymphocyte responsiveness to acetylcholine receptor (AChR) in rats with experimental autoimmune myasthenia gravis (EAMG). Rats were immunized with purified torpedo AChR. At intervals of one to eight weeks later, lymphocytes from the lymph nodes and spleen were cultured with purified torpedo AChR and rat muscle extract containing AChR. Lymphocyte responsiveness (stimulation index) was determined from uptake of 3H-labeled thymidine by the cultured cells. The response of lymphocytes to torpedo antigen began earlier and rose more rapidly than that to the homologous (rat) antigen. Lymph node cells responded more promptly than spleen lymphocytes. The stimulation indexes peaked at four to six weeks while antibodies to both antigen continued to rise. Delineation of this pattern of lymphocyte responsiveness sheds further light on the pathogenesis of the autoimmune response in EAMG and will be useful in the future design of immunotherapeutic strategies.

Further studies on aminopeptidases of rat muscle

1. I. Four aminopeptidase peaks (pH 7.5) were obtained from rat muscle extract on DEAE Sephadex. 2. 2. All showed little or no inhibition with 1 mM puromycin. 3. 3. Peak II hydrolysed alanine 2-naphthylamide at a much faster rate than other 2-naphthylamides. Peak III hydrolysed arginine 2-naphthylamide, with little or no activity on lcucine- or alanine-2-naphthylamides. Peak IV had broad specificity, methionine 2-naphthylamide being most rapidly split. Peak IV but not II readily hydrolysed the 7-amino-4-methylcournarin derivative of alanine. 4. 4. Peak II and IV were inhibited by Cu 2+, Zn 2+ and Co 2+ (10 μm or less), and II by similar concentrations of Mn 2+. Peak II was inhibited by l mM Mg 2+ and stimulated by l mM Ca 2+. 5. 5. Molecular weights (gel filtration) were: II. 50,000; III. 66,000; IV, 309,000.

Iron availability from meat.

1. The distribution of radioactive iron in 59Fe-labelled rat muscle extract was determined using gel filtration. This showed that most (approximately 70%) of the radioactivity was associated with the heamatin compounds; myoglobin and haemoglobin. 2. Raw beef and freeze-dried rat muscle were digested in vitro, under simulated physiological conditions, and after centrifugation the supernatants fractionated by gel filtration. The soluble products were haematin Fe complexes of molecular weight above 10,000 and non-haematin Fe compounds of molecular weight below 6000, the major products being the non-haematin Fe complexes. The soluble compounds were also separated by dialysis and, in rat muscle, it was found that the low-molecular-weight non-haematin compounds accounted for more than 80% of the total soluble iron. 3. In vivo absorption studies with rats showed the Fe in a digested muscle dialysate to be more readily absorbed than that from an aqueous muscle extract which itself was more readily absorbed than the Fe from whole blood. 4. It may not, therefore, be the haemoproteins per se which are responsible for the high availability of Fe in meat, but rather the nature of their degradation products, formed by digestion within the meat environment.

Separation of rat muscle aminopeptidases

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.

Enzym-Elektrophorese in Einschluß-Polymerisaten des Acrylamids. A. Amylasen, Phosphorylasen

Amylases of bacteria, barley, sweet potatoes, and white potatoes were separated by electrophoresis in polyacrylamide gels containing soluble starch. After incubation in acetate buffer and staining in iodine solution, the zones of enzyme activity remained unstained. The zones of crystalline α-amylase (bacillus subtilis) and of crude α-amylase (bacteriae) were found to be identical. The main zone of the crystalline β-amylase (lpomoea batatas) was identical with the zone of crude β-amylase (barley) and with the amylase zone of the potato (solanum tuberosum) extracts. Using 5 mm slots, 1 ng of amylase is traceable. Crystalline β-amylase yielded, in addition to the main zone, a minor zone nearer to the anode. The α-amylases had three zones of different properties. Phosphorylases were demonstrated as starch synthesizing and as starch degrading enzymes by making use of the reaction: “glycogen +glucose-1-phosphat⇌starch +phosphate”. Glycogen was polymerized into the gel to demonstrate the synthesis of starch, and incubation was carried out with glucose-1-phosphate. The starch formed was stained with iodine. The extracts of freshly harvested tubers yielded four zones, whereas those of stored tubers yielded two zones, and leaves and sprouts each showed one zone. - To observe starch degradation by phosphorylases, starch was polymerized into the polyacrylamide, and the gels were incubated in phosphate buffer. After treatment with iodine, an unstained zone was observed which corresponded to the most intense phosphorlase zone (close to the anode) in the glycogen gel. This zone was more intense in the tuber extracts than in leaves and sprouts. Pig heart extract contained the phosphorylase in the b form. Three zones have been observed. - Extracts of rat muscle and heart yielded phosphorylases which corresponded in their migration rate and in their requirement for adenosine-5′-monophosphate to the b form of pig heart phosphorylase. The same was true for the migration rate of liver phosphorylase. Crystalline α-phosphorylase of rabbit muscle required the addition of cystein to the electrophoresis buffer during separation. Two zones of activity could be demonstrated.