Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection.

The molecular basis of skeletal muscle weakness in a mouse model of inflammatory myopathy.

OBJECTIVE: It is generally believed that muscle weakness in patients with polymyositis and dermatomyositis is due to autoimmune and inflammatory processes. However, it has been observed that there is a poor correlation between the suppression of inflammation and a recovery of muscle function in these patients. This study was undertaken to examine whether nonimmune mechanisms also contribute to muscle weakness. In particular, it has been suggested that an acquired deficiency of AMP deaminase 1 (AMPD1) may be responsible for muscle weakness in myositis. METHODS: We performed comprehensive functional, behavioral, histologic, molecular, enzymatic, and metabolic assessments before and after the onset of inflammation in a class I major histocompatibility complex (MHC)-transgenic mouse model of autoimmune inflammatory myositis. RESULTS: Muscle weakness and metabolic disturbances were detectable in the mice prior to the appearance of infiltrating mononuclear cells. Force contraction analysis of muscle function revealed that weakness was correlated with AMPD1 expression and was myositis specific. Decreasing AMPD1 expression resulted in decreased muscle strength in healthy mice. Fiber typing suggested that fast-twitch muscles were converted to slow-twitch muscles as myositis progressed, and microarray results indicated that AMPD1 and other purine nucleotide pathway genes were suppressed, along with genes essential to glycolysis. CONCLUSION: These data suggest that an AMPD1 deficiency is acquired prior to overt muscle inflammation and is responsible, at least in part, for the muscle weakness that occurs in the mouse model of myositis. AMPD1 is therefore a potential therapeutic target in myositis.

Isozymes of rat AMP deaminase.

Three AMP deaminase isozymes (EC 3.5.4.6 AMP aminohydrolase) were purified from rat heart, kidney and muscle. These enzyme preparations contained only the required isozyme. Antisera to individual isozymes were prepared and immunological relationships were tested. There was no cross-reactivity as tested by precipitation experiments. The antisera precipitated only the corresponding isozyme and there was no effect on other isozymes. These isozymes were also different in Km values for AMP and in substrate specificity. From the present studies, combined with previous results, it seems clear that the heart, kidney and muscle enzymes are different basic types. It is proposed that the muscle enzyme be designated as AMP deaminase A; the enzyme in kidney and liver, AMP deaminase B; the enzyme in heart, AMP deaminase C. Brain extracts contained five isozymes; two parent isozymes (B and C) and presumably their three hybrids.

Immunologic evidence for three isoforms of AMP deaminase (AMPD) in mature skeletal muscle.

Four rabbit polyclonal antisera to purified AMP deaminase (AMPD) isozymes were used to precipitate homogenate AMPD activity from dissected gracilis, soleus and gastrocnemius muscles of the cat, rabbit, rat, mouse, Rhesus monkey, human and toad. The antisera were also tested against other unusual muscles: autonomically innervated striated muscle of the upper esophagus (UEM), skeletal muscle of patients with myo-AMPD deficiency and extraocular muscles (EOM) of humans and Rhesus monkeys. The reference antiserum, M, prepared against human psoas muscle AMPD, precipitated > 90% AMPD from all primate skeletal muscles tested, and from type-2 muscles of all mammals tested, but < 75% from cat and rodent soleus, toad gastrocnemius and primate UEM, EOM and myo-AMPD deficient muscles. Thus, a second isozyme was clearly indicated. Antibody B, against rat liver and kidney AMPD, had no effect with any muscle specimen. Antibody C, against rat heart AMPD, produced additive precipitation of AMPD from soleus of rat and mouse, while antibody E1, against human red cell (and heart) AMPD, produced additive AMPD precipitation from toad gastrocnemius, cat soleus and muscles of several AMPD-deficient humans. A second AMPD isozyme thus accounted for as much as 25% of total activity in some animal red muscles, but no more than 5% in human mixed muscles. At least one more isozyme is needed to account for muscle AMPD unreactive with all antibodies tested in rabbit soleus, toad gastrocnemius and primate UEM and EOM. A list is appended of the approximate AMPD activity in various human cells and tissues.

AMP-deaminases from chicken and rabbit muscle: partial primary sequences of homologous 17-kDa CNBr fragments: autorecognition by rabbit anti-[chicken AMPD].

The apparent size (87.5 kDa) of the major polypeptide in freshly isolated chicken muscle AMP deaminase (AMPD.M) was comparable with that predicted from the sequences of the genes for the major muscle isoforms from human and rat. The size of the subunit of AMP deaminase from chicken muscle is indistinguishable from that of the rabbit enzyme. The peptide profiles of cyanogen bromide digests of AMPD.M from chicken and rabbit share a 17-kDa fragment, representing approximately 20% of the intact subunits of these enzymes. The first 25 residues of these fragments are 88.5% identical; the rabbit and chicken segments are greater than 92% and 84% identical, respectively, to the sequences predicted for residues 310-335 for AMPD.M from human and rat. Polyclonal rabbit antisera directed against AMPD.M from chicken breast recognize the full-length AMPD.M polypeptides on immunoblots of extracts of both avian and rabbit muscle, including an antiserum from the rabbit in which the antibody was prepared. The 17-kDa fragments, derived by incomplete cleavage of highly conserved internal segments of the deaminase subunit, share epitopes involved in the autorecognition of rabbit AMPD.M by rabbit polyclonal antibodies directed against the avian AMPD.M.

Comparative immunologic and kinetic evaluation of AMP-deaminase isolated from normal human liver and hepatocellular carcinoma (HCC).

In the present paper physico-chemical properties of AMP-deaminase purified from human liver neoplasm-hepatocellular carcinoma (HCC) were investigated and compared with these obtained for the enzyme from normal, unaffected tissue.

Myoadenylate deaminase deficiency: primary and secondary types.

Myoadenylate deaminase deficiency, the most common of the known enzyme deficits of muscle, appears to occur in two forms. The primary type seems to be inherited as a complete gene block in an autosomal recessive pattern. Although occasionally diagnosed in infancy, when muscle biopsy is performed on a hypotonic but normoreflexic child, the deficiency is usually not symptomatic until adult or middle age, when muscle cramping and exercise intolerance develop. The skeletal muscle isozyme is immunologically, and presumably genetically, unique, and these patients have normal levels of adenylate deaminase in their other cells and tissues. A presumptive diagnosis can usually be made by an ischemic forearm exercise test, which shows a negligible increase in blood ammonia, despite a normal rise in lactate. Despite the absence of more than 99% of normal adenylate deaminase activity, the muscle biopsy shows no anatomic pathology, and other enzymes are at normal levels. These patients do not suffer progressive disease, and should be reassured and encouraged to maintain physical activity. The heterozygous state is probably asymptomatic, except, perhaps, on extreme exercise, but may be associated with an increased incidence of malignant hyperthermia susceptibility. Since the gene defect is not rare, it is not surprising that some cases of the deficiency will be coincidentally associated with other neuromuscular disease. However, there is also a secondary form of myoadenylate deaminase deficiency, consequent to muscle damage from other disease. In this form, the residual activity is higher (1-10% of normal), may present rare foci of positive stain in the section, and reacts normally with antibody to the muscle isozyme. Other muscle enzymes are also depleted, although not as severely, and the prognosis in such cases is dictated by the primary disease. Since the heterozygous state is common, these patients might have been carriers whose adenylate deaminase levels have been lowered to the deficient category by the advent of other neuromuscular disease.

Kinetic and immunologic evidence for a complete gene block in myoadenylate deaminase deficiency.

Muscle biopsies from patients with myoadenylate deaminase deficiency (mADD) have been evaluated kinetically and immunologically to ascertain the origin of residual enzyme activity. Kinetic evaluation employed 5 mM AMPS/1 mM AMP ratios, which were 0.7-0.8 for the human muscle isozyme, but 0-0.25 for the isozyme(s) of all other human blood cells and tissues examined. Of 14 control biopsies, 13 showed a ratio greater than 0.60 (one gave 0.47) regardless of the enzyme specific activity, while all 14 mADD biopsies showed a ratio less than 0.24, suggesting that a fetal muscle isozyme and/or blood cell isozyme were responsible for the residual activity. Confirmation was provided by rabbit antisera to purified human muscle AMP deaminase. These antisera fully precipitate the isozyme from crude human muscle biopsy homogenates, regardless of fiber-type composition, and cross-react effectively with the muscle isozyme of Rhesus monkeys and thoroughbred horses, but are inactive toward the isozymes of all other human blood cells and tissues examined. Of 18 mADD homogenates tested, 14 showed less than 20% reactivity with the antisera, at levels that precipitated 10 x more enzyme in control specimens. The residual activity in most cases of mADD must therefore arise from some source other than normal AMP deaminase. To evaluate the possibility of a single common determinant, 9 mADD homogenates were tested for soluble immune complexes. Seven of the 9 then showed 20-42% reactivity, suggesting that part of their residual activity may be due to an isozyme sharing one antigenic determinant with the normal muscle isozyme. Competitive antigen binding was used to assess whether catalytically inactive AMP deaminase was present in mADD. The method was demonstrated effective in identifying spontaneously inactivated purified enzyme and alkaline-inactivated crude enzyme. Nevertheless, homogenates from 17 mADD cases failed to produce more than 14% activation, under conditions in which 63-99% activation was expected. Triton X-100 extracts of homogenate residues of 11 mADD cases were also tested, in a search for insoluble antigen; none produced significant competition. The evidence thus indicates that most cases of mADD are due to a complete gene block, with total absence of all normal muscle AMP deaminase protein.

Influence on myoadenylate deaminase function in rat skeletal muscle after homologous and heterologous immunization with the purified enzyme.

The aim of the study was to shed some light on the possibility of anti-MAD autoantibodies in cases of rhabdomyolysis or microtraumatic muscular damage causing the symptoms of the acquired myoadenylate deaminase deficiency. Therefore a homologous and a heterologous immunization of rats was carried out with myoadenylate deaminase (MAD; EC 3.5.4.6.) isolated from rat white gastrocnemius muscle (type IIb) and rabbit skeletal muscle. Antibody response to homologous and heterologous antigens was quantified by ELISA and by the inhibition of MAD activity in vitro. Anti-MAD IgG antibodies obtained by homologous immunization caused an inhibition rate of 80% when using MAD from rat white gastrocnemius muscle. Furthermore, the function of MAD in homologously and heterologously immunized rats was investigated after maximal swimming exercise in comparison to non-immunized animals by determination of muscle ammonia and by HPLC detection of inosine 5'-monophosphate (IMP) in skeletal muscle tissues. Both IMP and ammonia accumulation were significantly reduced in type IIb muscle fibers after homologous immunization of rats compared to non-immunized controls (n = 7), whereas the concentration of muscle lactate showed no differences. According to these results we propose that by homologous immunization with purified MAD, anti-MAD IgG impairs the function of cytoplasmic MAD during vigorous exercise in vivo.

Treatment of experimental autoimmune encephalomyelitis with adenylate deaminase from Penicillium lanoso-viride.

The effect of intramuscularly administered immunomodulator, adenylate deaminase (E.C. 3.5.4.6), from Penicillium lanoso-viride on the clinical score of acute experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease, was examined by inoculation of guinea pigs with rabbit brain and spinal cord homogenate (encephalitogen) and complete Freund's adjuvant. Adenylate deaminase (ADA) was effective in delaying the onset of clinical disease. ADA inhibited the severity of EAE. There was a significant decrease in clinical signs. A decrease in the number of morbid and dead animals was observed. Of ADA treated animals, 50-80% developed no clinical manifestations of EAE. The optimal version of treatment was a single preventive injection of ADA 1 day before the sensitization and then every second day after immunization for 20 days. ADA treatment of immunized animals diminished the activity of 2', 3'-cyclic nucleotide 3'-phosphodiesterase in the cerebrospinal fluid, as well the amount of complement fixing antiencephalitogenic antibodies in the blood serum. The mechanism of ADA cerebroprotective action is discussed. Significant skin-allergic cross-reaction of delayed-type hypersensitivity between ADA and encephalitogen was observed.

Isolation and regulation of piglet cardiac AMP deaminase.

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a Km of 2 mM for 5'-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the Km of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD+, whereas, the Km was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in Vmax with no change in Km. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.