Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase

A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.     

Fine specificity of antibodies to the synthetic polypeptide poly(L-tyrosine, L-glutamic acid)-poly(DL-alanine)--poly(L-lysine) and its ordered analogs as followed by solid-phase radioimmunoassay

The fine specificity of antibodies against (T,G)-A--L and its ordered analogs (T-T-G-G)-A--L and (T-G-T-G)-A--L was studied. Fifty percent of the antibodies against (T,G)-A--L are directed toward the T-T-G-G determinants and 19% against T-G-T-G-like determinants. The rest of the antibody response to (T,G)-A--L is directed against determinants which exist in (T,G)-A--L but are not cross-reactive with either T-T-G-G- or T-G-T-G-like determinants. Although (T-T-G-G)-A--L and (T-G-T-G)-A--L differ only in the sequence of tyrosine and glutamic acid in their side chains, no crossreactivity was observed between antibodies toward the two ordered polypeptide antigens.     

Thymosin beta arg10, a major variant of thymosin beta 10 in rabbit tissues

Two homologous peptides, designated thymosin beta 4 and thymosin beta 10, respectively, have been shown to be widely distributed in mammalian cells and tissues (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576; S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker, (1983) Arch. Biochem. Biophys. 225, 407-413). In the rabbit, thymosin beta 4 is replaced by a variant, thymosin beta ala4, that contains alanine in place of serine at the blocked NH2-terminus. It is reported that in rabbit tissues thymosin beta 10 is also replaced by a variant, designated thymosin beta arg10, that contains an additional amino acid, arginine, inserted following lysine-38. The rabbit tissues analyzed also differ from those of other mammals in the relative quantities of thymosin beta ala4 and beta arg10, which are nearly equal, compared to tissues from other mammals where the quantities of thymosin beta 10 are only one-third to one-tenth those of thymosin beta 4.     

The inhibition of acetylcholinesterase by arsenite and fluoride

The effect of fluoride on the rate of reaction of acetylcholinesterase with arsenite, on the rate of dissociation of the enzyme-arsenite complex, and on the equilibrium between enzyme and arsenite was studied. Fluoride decreases the rate of the reaction between acetylcholinesterase and arsenite and changes the apparent equilibrium dissociation constant between the enzyme and arsenite, but even at concentrations as high as 0.2 M has no effect on the rate of dissociation of the enzyme-arsenite complex. The binding of fluoride and arsenite with the enzyme is highly anticooperative and may well be mutually exclusive. These results are consistent with a model in which the binding sites overlap and in which the same functional groups are involved.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.     

Biosynthesis of alpha-galactosidase A in cultured Chang liver cells

An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.     

Selective loss of mitochondrial genome can be caused by certain unsaturated fatty acids

Various unsaturated fatty acids had different effectiveness for maintaining the continued replication of functional mitochondria in an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae (KD115). Certain isomers of octadecenoic acid (i.e., cis-9) and eicosatrienoic acid (i.e.,cis-8,11,14) permitted continued replication of mitochondria and provided cultures that contained only 4 to 5% cells that formed petite colonies. On the other hand, cultures grown with cis-12- or cis-13-octadecenoic acid or cis-11,14,17-eicosatrienoic acid, produced a 12- to 16-fold greater frequency of petite mutants (50-60%) after 8 to 10 generations of growth. The production of the petite mutants occurred despite adequate incorporation of these unsaturated fatty acids into cellular phospholipids and an apparently normal ability to undergo the initial steps in the induction of cellular respiration. The evidence suggests that some cellular processes necessary for continued mitochondrial replication depend on the structural features of the fatty acyl chains as well as the overall content of unsaturated fatty acids in membrane phospholipids. Impairment of that process by certain inadequate fatty acids or by an inadequate supply of a suitable fatty acid leads to a permanent loss of the mitochondrial genome from the cells of subsequent generations.     

Calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction by decreasing the apparent binding affinity of heparin for thrombin

The present study has shown that calcium inhibits the heparin-catalyzed antithrombin III/thrombin reaction. The initial rate of thrombin (4.0 nM) inhibition by antithrombin III (200 nM) in the presence of heparin (2.5 ng/ml) decreased from 3.6 nM/min (in the absence of calcium) to 0.12 nM/min in the presence of 10 mM calcium. In the absence of heparin, the initial rate of thrombin inhibition by antithrombin III was not affected by calcium. The heparin-catalyzed antithrombin III/thrombin reaction is described by the general rate equation for a random-order, bireactant, enzyme-catalyzed reaction (M. J. Griffith (1982) J. Biol. Chem. 257, 13899-13902). As such, the reaction is saturable with respect to both thrombin and antithrombin III. The apparent kinetic parameters for the heparin-catalyzed antithrombin III/thrombin reaction were determined in the presence and absence of calcium. The apparent heparin/antithrombin III dissociation constant values were not measurably different in the presence of 0, 1.0, and 3.0 mM calcium. The apparent heparin/thrombin dissociation constant value increased from 7.0 nM, in the absence of calcium, to 10 and 30 nM in the presence of 1.0 and 3.0 mM calcium, respectively. The maximum reaction velocity, at saturation with respect to both proteins, was not affected by calcium. It is concluded that calcium binds to functional groups within the heparin molecule which are required for thrombin binding.     

Elucidation of a minimal immunoreactive site of vertebrate calmodulin

The heptapeptide AsnTyrGluGluPheValGlnNH₂ corresponding to residues 137–143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem.256, 4205–4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127–144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only sevenresidue segment in the 135–145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.