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Receptor for activated C-kinase 1 (RACK1) is an intracellular scaffolding protein involved in a multitude of signalling pathways. The cytoskeleton is fundamental for intracellular cell signalling as it forms an interconnected network of regulatory proteins. Here, spectrin is a central component as it forms the actin.spectrin network that serves as docking surfaces for cellular components. The interaction between RACK1 and components of spectrin, the single spectrin repeats R16, R17 and the double spectrin repeat R1617 from the α-spectrin chain were investigated by biosensor technology and docking analysis. RACK1 associated only weakly to R16 (KD = 1.0 ± 0.5 × 10-6 M), about 20 times stronger to R1617 (KD = 5.3 ± 0.7 × 10-8M) and 100 times stronger to R17 (KD = 0.9 ± 0.3 × 10-8 M). Docking analysis showed that while R16 alone preferentially docked with its B-helix, R17 docked through its A-helix and BC loop. The double repeat and RACK1 mainly formed two different complex conformations. R1617 docked tangentially to the N/C-terminal of RACK1 or radially along a groove on the outer surface of RACK1. These configurations could account for the slight increase in entropic and the decrease in enthalpic interactions for the R1617.RACK1 interaction, compared with the interactions of RACK1 to the two single repeats. Our results suggest a mode of interaction that allows spectrin to attach to the N/C part of RACK through the inter-helical AB and BC loops and adopt a multitude of configurations in between the two limiting configurations. [ABSTRACT FROM AUTHOR]

The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/a-pyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3′-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5′ ends of the nicks, indicating that the nicks possessed a 5′-phosphate group; 5′- and 3′-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified φX174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions. [ABSTRACT FROM AUTHOR]

The kinetics of T4 polynucleotide ligase has been investigated at pH 8, 20 °C and using the doublestranded DNA substrate (dA)n · [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 µM and that of ATP 100 µM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32Plabelled (dA)n · [(dT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70 mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents. [ABSTRACT FROM AUTHOR]

A radioactive soluble model protein for studies of protein digestion, absorption and amino acid (AA) metabolism in larval fish was successfully produced in Atlantic salmon by oral administration of uniformly [U]-14C-labelled amino acids followed by blood sample withdrawal (48 h post-administration) and purification. The salmon serum protein (14C-SSP) was characterised in terms of the protein composition and specific activity of its amino acids. Most radioactivity was found in the three most abundant serum proteins, which had apparent molecular weights of 65, 75 and 120 kDa, respectively, of which labelling was found in all the amino acid residues of the SSP that were analysed. The digestibility of the 14C-SSP was tested by in vivo tube feeding using early stages of Atlantic halibut and was found to be more efficiently digested and utilised than the 14C-methylated bovine serum albumin (14C-BSA) that has been used in previous studies. This supports the notion that proteins labelled by 14C-methylation are not suitable as model proteins in metabolic studies due to modification of their 14C-methylated lysine residues. Further studies on the 14C-SSP demonstrated a digestibility of 59±13% in juvenile halibut, while at the pre-metamorphic stage, it was only 25±13%. This supports the hypothesis that there is a significant improvement in the ability to digest and utilise dietary proteins as the digestive system becomes fully developed, including a functional (acid-producing) stomach. [Copyright &y& Elsevier]