Effect of phosphorylation by cyclic AMP-dependent protein kinase on the smooth muscle actomyosin Mg2+-ATPase stimulatory activity of fodrin

Fodrin, a non-erythrocyte spectrin-like protein, has been purified from bovine brain and found to be phosphorylated by the cyclic AMP-dependent protein kinase with a maximal stoichiometry of 1.02 +/- 0.06 mol of phosphate/mol of fodrin dimer (n = 4). This phosphorylation was not affected by the presence of actin and calmodulin. The phosphorylation of fodrin was found to occur exclusively at serine residues on the beta subunit. Two-dimensional thin layer electrophoresis and chromatography of a tryptic digest of phosphorylated fodrin showed one major phosphopeptide and a few minor ones. We have previously reported that nonphosphorylated fodrin is capable of stimulating the smooth muscle actomyosin Mg2+-ATPase by 50-70% under a well-defined set of conditions such as a critical fodrin concentration and an optimal preincubation time (Wang, C., Ngai, P.K., Walsh, M.P., and Wang, J.H. (1987) Biochemistry 24, 1110-1117). We now report that phosphorylation of fodrin completely eliminates this stimulatory effect. However, phosphorylation of fodrin was able to compete with nonphosphorylated fodrin to result in the abolition of the stimulatory effect. Similarly, nonphosphorylated fodrin could overcome the inhibitory effect created by phosphorylated fodrin. The present results support the suggestion that the stimulation of the smooth muscle actomyosin Mg2+-ATPase by fodrin may be a physiological phenomenon and cyclic AMP may serve as a regulator for this effect.     

Chorismate aminations: partial purification of Escherichia coli PABA synthase and mechanistic comparison with anthranilate synthase

Chorismate is converted by regiospecific amination/aromatization sequences to o-aminobenzoate and p-aminobenzoate (PABA) by anthranilate synthase (AS) and PABA synthase (PABS), respectively. We report here the first partial purification of the large subunit of Escherichia coli PABA synthase, previously reported to be quantitatively inactivated in purification attempts. The subunit encoded by the pabB gene was overexpressed from a T7 promoter and purified 9-fold to 25-30% homogeneity. The pabB subunit appears unusually sensitive to inactivation by glycerol so this cosolvent is contraindicated. The Km for chorismate is 42 microM in the ammonia-dependent conversion to PABA, and we estimate a turnover number of 2.6 min-1. A variety of chorismate analogues have been prepared and examined. Of these compounds, cycloheptadienyl analogue 11 has been found to be the most potent inhibitor of Serratia marcescens anthranilate synthase (Ki = 30 microM for an RS mixture) and of the E. coli pabB subunit of PABA synthase (Ki = 226 microM). Modifications in the substituents at C-3 [enolpyruyl ether, (R)- or (S)-lactyl ether, glycolyl ether] or C-4 (O-methyl) of chorismate lead to alternate substrates. The Vmax values for (R)- and (S)-lactyl ethers are down 10-20-fold for each enzyme, and V/K analyses show the (S)-lactyl chorismate analogue to be preferred by 12/1 over (R)-lactyl for anthranilate synthase while a 3/1 preference was observed for (R)-/(S)-lactyl analogues by PABA synthase. The glycolyl ether analogue of chorismate shows 15% Vmax vs. chorismate for anthranilate synthase but is actually a faster substrate (140%) than chorismate with PABA synthase, suggesting the elimination/aromatization step from an aminocyclohexadienyl species may be rate limiting with AS but not with PABS. Indeed, studies with (R)-lactyl analogue 14 and anthranilate synthase led to accumulation of an intermediate, isolable by high-performance liquid chromatography and characterized by NMR and UV-visible spectroscopy as 6-amino-5-[(1-carboxyethyl)oxy]-1,3-cyclohexadiene-1-carboxylic acid (17). This is the anticipated intermediate predicted by our previous work with conversion of synthetic trans-6-amino-5-[(1-carboxyethenyl)oxy]-1,3-cyclohexadiene-1-carbo xylic acid (2) to anthranilate by the enzyme. Compound 17 is quantitatively converted to anthranilate on reincubation with enzyme, but at a 1.3-10-fold lower Vmax than starting lactyl substrate 14 under the conditions investigated; the basis for this kinetic variation is not yet determined.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.     

Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.     

Influence of cyanobacterial diet on Asplanchna predation risk in Brachionus calyciflorus

Asplanchna sylvestrii does not discriminate between groups of Brachionus calyciflorus fed either the cyanobacterium Anabaena flos-aquae or a control diet of Euglena gracilis. We based our analysis on the observed probabilities of attack, capture and ingestion during encounters between predator and prey. While A. sylvestrii was very sensitive to brachionid size, we found no significant affects of prey diet on predatory behavior. Thus, cyanobacterial diet did not influence the short-term predation risk of B. calyciflorus exposed to an effective predator. On the other hand, matched cohorts of A. sylvestrii fed B. calyciflorus cultured on the cyanobacterium reproduced more slowly than those fed the same prey cultured on the control food. With prolonged sympatry, therefore, the long-term risk of Asplanchna predation may be reduced for Brachionus by the latter's consumption of cyanobacteria.     

Alternative splicing generates a secreted form of N-CAM in muscle and brain

A number of different membrane associated isoforms of the neural cell adhesion molecule (N-CAM) have previously been identified. Here the structure of a novel secreted isoform of N-CAM is established by analysis of a cDNA corresponding to an N-CAM mRNA from human skeletal muscle. The mRNA incorporates a novel sequence block into the extracellular domain, which introduces an in-frame stop codon and thus prematurely terminates the coding sequence, generating a truncated N-CAM polypeptide. Analysis of genomic clones indicates that the inserted sequence is present as a discrete exon within the human N-CAM gene, and Northern analysis shows it to be associated specifically with a 5.2 kb mRNA species from skeletal muscle and brain. Stable transfectants expressing the secreted isoform accumulate it in the cytoplasm and release it to the culture medium. In contrast, cells transfected with cDNA encoding lipid-tailed N-CAM express it predominantly at the cell surface. The existence of a secreted isoform may further expand the spectrum of N-CAM function beyond its known involvement in intercellular adhesion to extracellular matrix interactions.     

The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression.

The inhibitor protein (PKI) of the cAMP-dependent protein kinase was first characterized from rabbit skeletal muscle. More recently a form of PKI was isolated and cloned from rat testis which shares relatively limited amino acid sequence with the rabbit skeletal muscle form. We have now isolated a cDNA from rat brain which encodes a protein corresponding to the rabbit skeletal muscle PKI. This establishes the presence of the "skeletal muscle" and "testis" proteins in the same species and therefore that they clearly represent distinct isoforms. We have also demonstrated that the isoform from testis, like the skeletal muscle isoform, is specific for the cAMP-dependent protein kinase and that it is able to inhibit this enzyme when expressed in cultured JEG-3 cells. Both forms contain the five specific amino acid recognition determinants which have been shown to be required for high affinity binding to the protein kinase catalytic site, although there is some noted lack of conservation of codons used for these residues. Overall, the two rat isoforms are only 41% identical at the amino acid level and 46% at the level of coding nucleotides. We propose that the rabbit skeletal muscle and rat testis forms be designated PKI alpha and PKI beta, respectively. Using Northern blot analysis, we have examined the tissue distribution of the two forms in the rat and their relative expression during development. In the adult rat, mRNA of the PKI alpha species is highest in muscle (both skeletal and cardiac) and brain (cortex and cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural and synthetic DNA elements with the CArG motif differ in expression and protein-binding properties.

DNA elements with the CC(A/T)6GG, or CArG, motif occur in promoters that are under different regulatory controls. CArG elements from the skeletal actin, c-fos, and myogenin genes were tested for their abilities to confer tissue-specific expression on reporter genes when the individual elements were situated immediately upstream from a TATA element. The c-fos CArG element, also referred to as the serum response element (SRE), conferred basal, constitutive expression on the test promoter. The CArG motif from the myogenin gene was inactive. The skeletal actin CArG motif functioned as a muscle regulatory element (MRE) in that basal expression was detected only in muscle cultures. Muscle-specific expression from the 28-bp MRE and the 2.3-kb skeletal actin promoter was trans repressed by the Fos and Jun proteins. The expression and factor-binding properties of a series of synthetic CArG elements were analyzed. Muscle-specific expression was conferred by perfect 28-bp palindromes on the left and right halves of the skeletal actin MRE. Chimeric elements of the skeletal actin MRE and the c-fos SRE differed in their expression properties. Muscle-specific expression was observed when the left half of the MRE was fused to the right half of the SRE. Constitutive expression was conferred by a chimera with the right half of the MRE fused to the left half of the SRE and by chimeras which exchanged the central CC(A/T)6GG sequences. At least three distinct proteins specifically bound to these CArG elements. The natural and synthetic CArG elements differed in their affinities for these proteins; however, muscle-specific expression could not be attributed to differences in the binding of a single protein. Furthermore, the MRE did not bind MyoD or the myogenin-E12 heterodimer, indicating that muscle-specific expression from this element does not involve a direct interaction with these helix-loop-helix proteins. These data demonstrate that the conserved CArG motifs form the core of a family of functionally different DNA regulatory elements that may contribute to the tissue-specific expression properties of their cognate promoters.