A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism.

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.

Increased circulating levels of ouabain-like factor in patients with asymptomatic left ventricular dysfunction.

BACKGROUND: Much evidence has been accumulated that human plasma contains digitalis-like factor(s) with Na/K ATPase inhibitor properties. Increased concentrations of ouabain-like factor (OLF) have been reported in patients with moderate to severe hypertension and in patients with overt congestive heart failure due to dilated cardiomyopathy. AIM: The presence of circulating OLF has not been investigated in borderline to mild hypertension or in the early stage of dilated cardiomyopathy. METHODS AND RESULTS: The study population consisted of 18 normal volunteers, 24 patients with borderline to mild hypertension, 47 patients with asymptomatic left ventricular dysfunction (ALVD) due to dilated cardiomyopathy and 26 patients with cardiac arrhythmias but normal left ventricular function. OLF values (pM ouabain equivalent) were assayed in extracted plasma, using a radioimmunoassay for ouabain. OLF was, respectively, 29.4+/-20.6 pM in normal controls, 39.1+/-23.8 pM in hypertensives, 35+/-18 pM in patients with cardiac arrhythmias, 52.3+/-25.8 pM in ALVD patients not treated with digoxin and 64.6+/-29.6 pM in ALVD patients treated with digoxin. Patients with ALVD, both treated and not treated with digoxin, had OLF significantly higher (P<0.05) than all the other groups. In patients with ALVD no correlation between OLF and left ventricular ejection fraction was observed. In the hypertensive group no correlation between OLF and both diastolic and systolic pressure was found. CONCLUSION: Increased concentrations of OLF were observed in patients with left ventricular dysfunction due to dilated cardiomyopathy, before the occurrence of overt heart failure, suggesting that OLF may be an early marker of the disease.

Detection of digitalis compounds using a surface plasmon resonance-based biosensor.

An automated surface plasmon resonance-based biosensor system has been used to detect endogenous and exogenous digitalis-like factors (EDLF) in the pmolar range in real time. EDLF was purified from umbilical cord blood. EDLF has been suggested to play a role in hypertension and in perinatal adaptation. Highly specific polyclonal anti-ouabain antibodies showed a high affinity binding capacity for ouabain, ouabagenin and strophantidin with an IC50 value of 5 x 10(-10) M, 7.0 x 10(-10) M and 2 x 10(-8) M, respectively. EDLF cross-reacted with antibodies and its concentration in plasma at IC50 was around 50 pmol ouabain equivalent. This study shows the potential usefulness of the biosensor technology for biomolecular interaction analysis. The features of this technology (fully automated, measured in real time, sharpened response) offer several advantages compared with a traditional immunoassay like radioimmunoassay (RIA) in the detection of digitalis compounds in human fluids.

Correlation between endogenous digoxin-like immunoreactivity and 3H-ouabain displacement on erythrocyte membranes in extracts of human plasma.

The existence of endogenous cardiac glycoside-like compounds and their property of being recognized by anti-digoxin antibodies is still a matter of controversy. In order to investigate this problem, endogenous digoxin-like immunoreactivity (measured by RIA) and digitalis-like radioreceptor activity (measured by displacement of 3H-ouabain from erythrocyte membranes) were assessed in plasma extracts of normal adults, pregnant women and newborns. These three groups were chosen because of their known widely different levels of digoxin-like immunoreactivity. Compared to adults, newborns and pregnant women had significantly higher levels not only of immunoreactivity but also of displacement of 3H-ouabain binding, the latter being due, according to Scatchard analysis, to a decrease of the affinity of ouabain to its cellular receptor rather than to its maximal binding capacity. Furthermore, immunoreactivity and binding displacement correlated significantly. Our data indicate that one (or more) compounds with cardiac glycoside-like properties (both immunological and at the receptor level) are present in the plasma of newborns and pregnant women, and confirm the idea that radioimmunological methods may be useful in studying endogenous inhibitors of the sodium pump.

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor.

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.

Further evidence for an endogenous digitalis-like compound in newborn and adult plasma detected by anti-ouabain antiserum.

It is widely but not unanimously accepted that one or more endogenous digitalis-like factors (EDLF) circulate in human plasma. In this paper we provide confirmatory evidence that newborn plasma contains a factor with immunological and biological properties similar to ouabain and demonstrate that this factor may be present also in the adult. In fact, we obtained in newborn and adult plasma extracts, identical HPLC elution profiles of ouabain-like immunoreactivity and 86Rb erythrocyte uptake inhibitory activity with a major peak corresponding to the retention time of ouabain. The fact that immunoreactivity and biological digitalis-like activity in the peak are due to an identical substance is strongly supported by the correlation between RIA and 86Rb uptake inhibitory values observed in the purified fractions. Finally, the strong correlation between immunoreactivity observed in plasma samples after simple SepPak C18 extraction and after additional HPLC suggests that less purified samples may be assayed for screening purposes. However, for a more quantitative assessment, this simple extraction method needs a subsequent HPLC purification for eliminating an overestimation of values due to cross-reacting impurities.

Evidence for an endogenous ouabain-like immunoreactive factor in human newborn plasma coeluted with ouabain on HPLC.

The identification in human plasma of ouabain as an endogenous digitalis-like factor (EDLF) claimed by Hamlyn et al. has recently been contradicted by two studies which failed to demonstrate endogenous ouabain-like immunoreactivity in HPLC fractions in which exogenous ouabain was eluted. In this paper we report the results obtained on the cross-reactivity with antiouabain antibodies of an EDLF purified by us from human newborn cord plasma. We found that this EDLF coeluted with ouabain on HPLC and cross-reacted both with rabbit anti-ouabain antiserum and with the purified antibodies, which excluded possible interferences due to antibodies directed against non-ouabain portions of the immunogen. Similar but not identical slopes of the ouabain and EDLF displacements curves were observed in all competition ELISA experiments. The inhibitory effect of EDLF on erythrocyte 86Rb uptake was reversed by antiouabain antiserum and antibodies. The concentration of EDLF in newborn plasma, in the four different purifications studied ranged from 30 to 380 pM ouabain equivalents (o.e.) by ELISA and from 100 to 300 pM o.e. by 86Rb uptake. Our data thus support the existence, in human newborn plasma, of a factor with both biological and immunological ouabain-like properties, although not necessarily identical to ouabain.

Evidence of marked digoxin-like immunoreactivity in the human adrenal cortex: results of an immunohistochemical study.

Several lines of evidence suggest the existence, in mammalian body fluids and tissue extracts, of an as yet unidentified endogenous digitalis-like factor with potential cross-immunoreactivity with digitalis. Using anti-digoxin antibodies, we found preliminary immunohistochemical evidence of digoxin-like immunoreactivity in several human tissues. Strong reactivity was found in the adrenal cortex, which may thus represent a site of production of this endogenous factor.

Cardiotoxic effects of adriamycin and mitochondrial oxidation in rat cardiac tissue.

Rats were subjected to chronic treatment with adriamycin (ADR). Significant alterations of ECG tracings were induced, starting from the third week of treatment. These alterations were related to mitochondrial damage of the heart tissue. A decrease of respiration rate in phosphorylating conditions was observed in isolated organelles over a period of three weeks. Meanwhile, adriamycinol (ADRol) concentration in heart extracts increased during chronic treatment.

A powerful inhibitor of guanine deaminase.

The synthesis of 9-(p-carbetoxyphenyl) guanine is reported. The assays carried out on guanine deaminase from rat and rabbit liver and pig brain show that this compound is a powerful inhibitor. The compound has a Ki = 5 micronM for the enzyme from pig brain. The use of the inhibitor for the synthesis of a specific adsorbent for guanine deaminase was studied.

beta 2-Microglobulin and other low molecular weight proteins in haemodiafiltrates and in haemofiltrates.

Uraemic patients underwent haemodialysis by cuprophan membrane, or haemodiafiltration by polyacrylonitrile membrane, or haemofiltration by acetate cellulose membrane. Plasma samples drawn before and after treatment with the dialyser were gel-filtered on Sephadex G-75. A comparison of the chromatograms showed that the treatment causes a removal of middle molecules, which is more consistent when haemodiafiltration and haemofiltration are used. Protein removal was examined in the fluids from the three dialysers. It was observed that proteins (300 +/- 45 mg and 410 +/- 60 mg respectively) were removed when polyacrylonitrile and acetate cellulose membranes were used, but not in the case of cuprophan membrane. beta 2-Microglobulin was the protein lost in the greatest amount.

Characterization of the carrier protein of digoxin-like immunoreactive substance in plasma.

The presence of an endogenous digoxin-like immunoreactive substance(s) (DLIS) has been reported in the plasma and urine of experimental animals and humans. This substance might have a role in arterial hypertension. Although the chemical structure of DLIS is at present unknown, several studies indicate that DLIS has a low molecular weight and is reversibly bound in serum to carrier proteins. We have investigated the carrier protein of DLIS in chromatographic studies, using neonate plasma eluted on a Sephadex G200 column. The chromatographic profile of digoxin-like immunoreactivity showed a major peak with a molecular weight of approximately 60,000 daltons and a smaller peak eluted after the salt region. When the major peak was chromatographed on a Sephadex G100 column only one single peak was obtained, which closely coincided with the elution peak of albumin. Chromatographic separation of neonate plasma on a Sephadex G25 revealed a major post-salt immunoreactive peak. When these fractions were incubated with purified human albumin and separated again on the Sephadex G25, a large immunoreactive peak corresponding to albumin was again found while the immunoreactivity after salt completely disappeared. Our data therefore strongly suggest that the endogenous digitalis-like factor is carried in plasma by albumin.

Purification of endogenous digitalis-like factor(s) from cord blood of neonate by immunoaffinity chromatography.

We have previously shown that antidigoxin antibodies may neutralize partially purified endogenous digitalis like factor(s) present in newborn (umbilical cord) plasma. We here report on the preparation of an immunoaffinity chromatographic system (high affinity digoxin-binding antibodies (Fab fragments) bound covalently to Sepharose) for the purification of endogenous digitalis like factor(s). Neonate plasma extract loses all its biological digitalis-like activity (erythrocyte 86Rb uptake inhibition) after absorption on Sepharose coupled to Fab fragments but not after absorption on uncoupled Sepharose. Endogenous digitalis like factor(s) absorbed to Sepharose coupled to Fab fragments can be eluted by methanol. Subsequent HPLC separation indicate that at least two molecular species with digitalis-like properties are retained by antibodies bound to Sepharose and can be recovered with methanol.

A general method of purification of adenosine deaminase by affinity chromatography.

Affinity chromatography has been used to purify adenosine deaminase from various sources: calf spleen, calf intestinal mucosa, chicken duodena and human erythrocytes. For this purpose a specific inhibitor, 9-(p-aminobenzyl) adenine, was synthesized and covalently joined to agarose. Adenosine deaminase is selectively retained by such an inhibitor-resin when highly impure solutions are chromatographed through it. After elution from the resin with guanylurea, a competitive inhibitor, the enzyme is homogeneous and can be recovered in yields of 80 percent or more and the same number of multiple forms of the enzyme is present in the purified preparation and in the crude extract.