Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

The ventilation, lactate and electromyographic thresholds during incremental exercise tests in normoxia, hypoxia and hyperoxia

These experiments examined the effect of hypoxia and hyperoxia on ventilation, lactate concentration and electromyographic activity during an incremental exercise test in order to determine if coincident chances in ventilation and electromyographic activity occur during an incremental exercise test, despite an enhancement or reduction of peripheral chemoreceptor activity. In addition, these experiments were completed to determine if electromyographic activity and ventilation are enhanced or reduced in response to the inspiration of oxygen-depleted and oxygen-enriched air, respectively. Seven subjects performed three incremental exercise tests, until volitional exhaustion was achieved, while inspiring air with a fractional concentration of oxygen of either 66%, 21% or 17%. In addition, another single subject completed two tests while inspiring air with a fractional concentration of either 17% or 21%. During the tests, ventilation, mixed expired oxygen and carbon dioxide, arterialized venous blood and the electromyographic activity from the vastus lateralis were sampled. From these values ventilation, electromyographic and lactate thresholds were detected during normoxia, hypoxia and hyperoxia. The results showed that although ventilation and lactate concentration were significantly less during hyperoxia as compared to normoxia or hypoxia, the carbon dioxide production values were not significantly different between the normoxic, hypoxic and hyperoxic conditions. For a particular condition, the time, carbon dioxide production and oxygen consumption values that corresponded to the ventilation and electromyographic thresholds were not significantly different, but the values corresponding to the lactate threshold were significantly less than those for the electromyographic and ventilation thresholds. Comparisons between the three conditions showed that the time, carbon dioxide production and oxyen consumption values corresponding to each of these thresholds were not significantly different. These findings have led us to conclude that the changes in lactate concentration observed during exercise may not be directly related to the fractional concentration of inspired oxygen, and that the peripheral chemoreceptors may not be the sole mediators of the first ventilatory threshold. It is suggested that this threshold may be mediated by an increase in neural activity originating from higher motor centers or the exercising limbs, induced in response to the need to progressively recruit fast twitch muscle fibers as exercise power output is increased and as individual muscle fibers begin to fatigue.     

Comparative analysis of multiple protein-sequence alignment methods [published erratum appears in Mol Biol Evol 1994 Sep;11(5):811]

We have analyzed a total of 12 different global and local multiple protein-sequence alignment methods. The purpose of this study is to evaluate each method's ability to correctly identify the ordered series of motifs found among all members of a given protein family. Four phylogenetically distributed sets of sequences from the hemoglobin, kinase, aspartic acid protease, and ribonuclease H protein families were used to test the methods. The performance of all 12 methods was affected by (1) the number of sequences in the test sets, (2) the degree of similarity among the sequences, and (3) the number of indels required to produce a multiple alignment. Global methods generally performed better than local methods in the detection of motif patterns.      

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

Mechanism-based inactivation of leukotriene A4 hydrolase/aminopeptidase by leukotriene A4. Mass spectrometric and kinetic characterization.

"Suicide" inactivation of leukotriene (LT) A4 hydrolase/aminopeptidase occurs via an irreversible mechanism-based process which is saturable, of pseudo firstorder, and dependent upon catalysis. Data obtained with either recombinant enzyme or enzyme purified from human leukocytes were similar. Apparent binding constants and inactivation rate constants are equivalent, compatible with a single type of substrate-enzyme complex which partitions between two fates, turnover and inactivation. Both catalytic functions are inactivated, consistent with an overlapping active site for this bifunctional enzyme. The partition ratio (turnover/inactivation) for the LTA4-enzyme complex is 129 +/- 16 for LTA4 hydrolase activity and 124 +/- 10 for aminopeptidase activity. The pH dependence for turnover and inactivation are indistinguishable with a maximum at pH 8. L-Proline p-nitroanilide, a weak substrate with a high Km for the aminopeptidase affords only partial protection against inactivation by LTA4. However, two potent competitive inhibitors, bestatin and captopril, protect both catalytic processes from inactivation, consistent with an active-site specificity for the suicide event. Electrospray ionization mass spectrometry indicates that the molecular weight of pure recombinant enzyme is 69,399 +/- 4 and that covalent modification accompanies catalysis, producing an LTA4:enzyme adduct with a molecular weight 69,717 +/- 4 and a 1:1 stoichiometry. In agreement with kinetic data, electrospray ionization mass spectrometry shows that bestatin inhibits the covalent modification of enzyme by LTA4 and that the extent of modification is proportional to the loss of enzymatic activity.