Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues

Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron.     

Isolation and characterization of folded fragments released by Staphylococcal aureus proteinase from the non-histone chromosomal protein HMG-1

HMG-1 was isolated from newborn calf thymus without exposure to overt denaturing conditions. The purified protein was digested under several solvent conditions with the proteinase (endoproteinase GluC) from Staphylococcus aureus strain V8. We found that the preferred site of attack by the enzyme on HMG-1 was influenced markedly by ionic strength and temperature. In 0.35 M NaCl/50 mM Tris-phosphate (pH 7.8) at 37 degrees C, cleavage near the junction between the A and B domains is predominant, as previously reported by Carballo et al. (EMBO J. 2 (1983) 1759-1764). However, in 50 mM Tris-phosphate (pH 7.8) lacking NaCl and at 0 degrees C, cleavage between the B and C domains strongly predominates. Three major products of the digestions were purified and characterized. The fragment consisting of domains B and C was found by circular dichroism to contain a substantial amount of helix. This re-emphasizes the importance of avoiding overt denaturing conditions when working with members of the HMG-1 family.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus.     

Evidence that noradrenaline increases pyruvate dehydrogenase activity and decreases acetyl-CoA carboxylase activity in rat interscapular brown adipose tissue in vivo.

The rate of fatty acid synthesis in interscapular brown adipose tissue of female cold-adapted rats, as measured by the incorporation of 3H from 3H2O into tissue lipid, was decreased by about 70% after injection of noradrenaline. There was a similar decrease in the activity of acetyl-CoA carboxylase. In contrast, the proportion of pyruvate dehydrogenase in its active non-phosphorylated form was greatly increased after injection of noradrenaline. This finding suggests that the oxidation of glucose may be important in noradrenaline-induced thermogenesis in rat brown adipose tissue.     

Beta-adrenergic agents increase the phosphorylation of phosphofructokinase in isolated rat epididymal white adipose tissue.

Pieces of rat epididymal adipose tissue were incubated in medium containing [32P]phosphate for 2 h to achieve steady-state labelling of intracellular phosphoproteins and then with or without hormones for a further 15 min. Phosphofructokinase was rapidly isolated from the tissue by use of either Blue Dextran-Sepharose chromatography or immunoprecipitation with antisera raised against phosphofructokinase purified from rat interscapular brown adipose tissue. Similar extents of incorporation of 32P into phosphofructokinase were measured by both techniques. Exposure of the tissue to adrenaline or the beta-agonist isoprenaline increased phosphorylation by about 5-fold (to about 1.4 mol of phosphate/mol of enzyme tetramer). No change in phosphorylation was detected with the alpha-agonist phenylephrine, but exposure to insulin resulted in an approx. 2-fold increase. The increased phosphorylation observed with isoprenaline was found to be associated with a decrease in the apparent Ka for fructose 2,6-bisphosphate similar to that observed on phosphorylation of phosphofructokinase purified from rat epididymal white adipose tissue with the catalytic subunit of cyclic AMP-dependent protein kinase. These results support the view [Sale & Denton (1985) Biochem. J. 232, 897-904] that an increase in cyclic AMP in adipose tissue may result in an increase in glycolysis through the phosphorylation of phosphofructokinase by cyclic AMP-dependent protein kinase.     

What makes wild rabbits drink?

Wild rabbits Oryctolagus cuniculus (L) introduced to Australia over a century ago successfully colonized diverse environments in a large part of the continent varying from arid desert, alps, to lush grasslands and coastline where water and salt may be either abundant or very scarce. Wild rabbits caught in Northern Victoria were studied under laboratory conditions, where they adapted to dry pelleted food and drank regularly water and a cafeteria of electrolyte solutions offered. Intracerebroventricular (IVT) infusion of angiotensin II (AII) in doses 10, 50 and 500 ng/h did not increase their water drinking, but increased salt appetite, although it was delayed one or more days after the beginning of AII infusion. IVT infusion of AII 500 ng/h for one day caused a halving in water intake and a tenfold increase in sodium excretion. These were followed by compensatory changes in water and 0.5 M NaCl intake on the consecutive days. IVT infusion of AII 50 ng/h for one day induced an increased urinary sodium excretion, a negative sodium balance which was not followed by an increased salt appetite. IVT infusion of AII 10 ng/h for five days caused a progressive increase in sodium excretion and salt appetite which were significant on the fourth day of infusion and both remained eight-ten times greater than control levels for three days after the cessation of infusion. Water intake was unchanged. IVT infusion of 0.3 M Na-CSF for two days reduced water and food intake, and caused a negative sodium balance on the second day of infusion which was not followed by increase in salt appetite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase.

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.