Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Studies on the protein-bound chondroitin sulphate of bovine cortical bone

A fraction containing chondroitin sulphate, isolated from bovine cortical bone under mild conditions, was separated by ion-exchange chromatography into three fractions with apparent homogeneity on electrophoresis and ultracentrifugation. Two of these appeared to consist of chondroitin sulphate bound to a glycoprotein ;core' that had similarities to the bone sialoprotein described previously. The differences in composition of the two fractions were considered to be due to variation in the number or lengths of the polysaccharide chains. The presence of xylose and the alkali-lability of the bond between protein and polysaccharide suggested the presence of a xylosylserine linkage. The third fraction had the properties of a relatively pure chondroitin sulphate which contained a small amount of peptide. These fractions differed considerably from the protein-polysaccharide complexes of epiphysial and other cartilages, and their relevance to the possible role of glycosaminoglycans is discussed.     

Some studies on the composition of bovine cortical-bone sialoprotein

1. An analysis of bovine bone sialoprotein, a homogeneous glycoprotein isolated from cortical bone, is presented. 2. Analytical results agree with earlier physical measurements indicating a molecular weight of about 23000. 3. Mild acid hydrolysis and treatment with neuraminidase showed that fucose and sialic acid occupy terminal positions on oligosaccharide chains. 4. Treatment of the sialic acid-free glycoprotein with beta-galactosidase showed that much of the galactose occupies a sub-terminal location in the intact glycoprotein. 5. The polypeptide chain is rich in aspartic acid, glutamic acid, serine, threonine and glycine, and has no detectable free terminal amino group. 6. Glycopeptides were studied after proteolytic digestion. 7. It is considered that the carbohydrate moiety is highly branched and is probably linked by an acid- and alkali-stable glycosylamine bond involving aspartic acid.     

Observations on bioluminescence in some deep-water anthozoans

The luminous responses to electrical stimulation of isolated polyps of 4 deep-water anthozoans are described. All show facilitatory responses and summation at stimulus frequencies > 2 s^–1. The responses of the gorgonian Acanella arbuscula comprise a slow summation of weak individual flashes. It is suggested that there is no fundamental difference between deep and shallow species, nor between the responses of scyphozoans, such as Pelagia and Atolla, and anthozoans, such as those described here. Both facilitated and decremental responses can be obtained from each of the 2 groups and the complexity of in vivo responses may be as much a reflection of selective pressures on the neural pathways as on the bioluminescent systems themselves.     

The carboxyl terminus of the smooth muscle myosin light chain kinase is expressed as an independent protein, telokin.

It has been proposed that the carboxyl terminus of the smooth muscle myosin light chain kinase is expressed as an independent protein. This protein has been purified from tissues and named telokin (Ito, M., Dabrowska, R., Guerriero, V., Jr., and Hartshorne, D. J. (1989) J. Biol. Chem. 264, 13971-13974). In this study we have isolated and characterized cDNA and genomic clones encoding telokin. Analysis of a genomic DNA clone suggests that the mRNA encoding telokin arises from a promoter which appears to be located within an intron of the smooth muscle myosin light chain kinase (MLCK) gene. This intron interrupts exons encoding the calmodulin binding domain of the kinase. The amino acid sequence deduced from the cDNA predicts that telokin is identical to the carboxyl-terminal 155 residues of the smooth muscle MLCK. Unlike the smooth muscle MLCK which is expressed in both smooth and non-muscle tissues, telokin is expressed in some smooth muscle tissues but has not been detected in aortic smooth muscle or in any non-muscle tissues.     

Basic residues are important for Ca2+/calmodulin binding and activation but not autoinhibition of rabbit skeletal muscle myosin light chain kinase

Several allosterically modulated protein kinases have been shown to be regulated by an autoinhibitory domain located within the kinase molecules. The inhibitory domain has been proposed to act as a "pseudosubstrate" inhibitor binding to the substrate binding site of the kinase, thereby blocking the binding of the enzyme's true substrate. In this report, site-directed mutagenesis has been used to further investigate the mechanism of activation of the inhibitory domain of rabbit skeletal muscle myosin light chain kinase. Basic residues within the pseudosubstrate domain (572-573, 577-579, 580-581), which are analogous to the important substrate determinants of the myosin light chain, were found not to be required in order to maintain the kinase in an inhibited state. Two groups of these residues (577-579 and 581-582) were, however, found to be important for high affinity calmodulin binding to the kinase. These data suggest that the autoinhibitory domain of myosin light chain kinase may not function by directly mimicking the light chain substrate.