Zeta-crystallin versus other members of the alcohol dehydrogenase super-family. Variability as a functional characteristic.

Species variability of the lens protein zeta-crystallin was correlated with those of alcohol dehydrogenases of classes I and III and sorbitol dehydrogenase in the same protein family. The extent of overall variability, nature of residues conserved, and patterns of segment variability, all fall within the limits typical of the 'variable' group of medium-chain alcohol dehydrogenases. This shows that zeta-crystallin is subject to restrictions similar to those of classical liver alcohol dehydrogenase and therefore derived from a metabolically active enzyme like other enzyme crystallins. Special residues at the active site, however, differ substantially, including an apparent lack of a zinc-binding site. This is compatible with altered functional properties and makes the spread within this medium-chain dehydrogenase family resemble the wide spread within the short-chain dehydrogenases. Schematic plotting is useful for illustrating the differences between 'variable' and 'constant' enzymes.

The presence of Z-helical conformation in DNA of the calf lens.

The purpose of this study was to reveal the presence of Z-helical conformation in normal crystalline lens DNA. Z-DNA antigen was prepared against poly(dG-dC).poly(dG-dC), which had been converted to the Z-helix conformation in high salt and then stabilized by bromination. Circular dichroism (CD) spectra confirmed the presence of left-handed Z-helix DNA. Antibodies to Z-DNA were raised in three rabbits immunized with brominated (Br-) poly(dG-dC).poly(dG-dC). These antibodies do not cross-react with polynucleotides in the B-helical form, but are specific to the left-handed Z-DNA conformation. DNA was isolated from three different regions of the calf lens. Anti-Z-DNA antisera, affinity purified IgG polyclonal anti-Z-DNA antibodies and monoclonal anti-Z-DNA antibodies were used as immunoprobes to detect the presence of S-DNA sequences. DNA from the cortex region of the lens reacted strongly with the anti-Z-DNA antibodies, but no binding could be observed in the DNA from the nucleus region. Digestion of lens DNA with DNase 1 dramatically decreased Z-DNA antibody binding, while RNase A and T1 treatment had no effect on Z-DNA immunoreactivity. This study has demonstrated that: (a) Z-DNA antibodies developed for our study can bind in high salt solutions (4M NaCl) to purified lens DNA sequences isolated from a variety of different calf lens cell types. By this criterion, lens DNA contains sequence determinants which may assume or are in the Z-helix conformation.

Detection of an enzyme activity which cleaves m7 guanine from m7 GMP in an extract of embryonic chick lens cells.

m7Guanine was cleaved from m7GMP by cytoplasmic enzyme activity in an extract prepared from embryonic chick lens cells. The appearance of m7-Guanine was proportional to the time and concentration of extract. m7-Guanine inhibited the reaction but neither guanine nor ribose 5-phosphate did. m7Guanine was not released from m7Guanosine. m7Guanine may be derived from m7GpppG mRNA cap by two enzymatic reactions with m7CMP as a product-substrate intermediate.

Differential synthesis of lens proteins in the presence of m7G(5')pppG and cleavage product m7GMP in an embryonic chick lens cell lysate.

The effect of m7GMP release from m7GMP-containing mRNA cap sequence m7GpppG by the embryonic chick lens m7GpppN-pyrophosphatase activity on the synthesis of lens proteins was examined in a newly developed homologous translation system derived from 15-day embryonic chick lenses. The synthesis of total lens polypeptides and delta-crystallin polypeptides, the major translation product, was inhibited 84% and 88%, respectively, by 0.5 mM m7GpppG; m7GMP (0.5 mM) inhibited total synthesis by 63% but was 33% less inhibitory toward delta-crystallin synthesis; GpppG and GMP were not inhibitors, m7GpppG inhibited met-tRNAfmet incorporation into 80S initiation complexes.

Cleavage of pm7G from mRNA 5' terminal cap structures by pyrophosphatase activity in embryonic chick lens cells.

The presence of pyrophosphatase activity in embryonic lens cells which cleaves pm7G and ppGm from m7G(5')pppGm was demonstrated. It was also found that m7G(5')pppG, but not G(5')pppG, was hydrolyzed, and conversion of m7GpppG to m7G*pppG, in which the 5-membered ring of the m7G moiety is open, abolished its hydrolysis. For the caps hydrolyzed, pm7G was released only in the presence of lens cellular fraction; pm7G inhibited cap hydrolysis.

Detection of methyltransferase activities which modify Gppp G to m7GpppGm in embryonic chick lens.

GpppG was modified to m7 GpppGm by a cytoplasmic extract, prepared from embryonic lens cells, in a reaction mixture which contained S-adenosyl-methionine as methyl group donor. The appearance of m7 GpppGm was a function of time and lens extract concentration. S-adenosyl-homocysteine inhibited both the m7G and Gm modification reactions. Analogues of GpppG, pG, ppG and pppG were relatively ineffective as substrates.

Synthesis of delta crystallin from embryonic chick lens messenger ribonucleoprotein complex.

A single, major 21 S messenger ribonucleoprotein (mRNP complex) was isolated and purified by sucrose gradient centrifugation after EDTA treatment of high salt washed polysomes from 15 day embryonic chick lenses. A 17 S mRNA was released from the 21 S subunit of delta crystallin. Similar results were obtained with the 17 S mRNA released from the 21 S mRNP complex.

Calf lens messenger ribonucleoprotein complexes. Characterization and comparison of template activity with corresponding mRNAs.

Lens messenger ribonucleoprotein complexes have been isolated from calf lens polysomes by sucrose gradient centrifugation after puromycin-induced dissociation. A 10 S mRNA was released from a 13 S messenger ribonucleoprotein complex and a 14 S mRNA from a 19 S messenger ribonucleoprotein complex. Two major protein components with molecular weights of approx. 64 000 and 40 000 were isolated from each of the messenger ribonucleoprotein complexes after RNAase digestion. Buoyant density determinations suggest that the messenger ribonucleoprotein complexes contain approximately one mol of each major protein species per mol mRNA. In contrast to lens mRNA, lens messenger ribonucleoproteins are poor templates for transcription with avian myeloblastosis virus reverse transcriptase. Similar results were also obtained with globin messenger ribonucleoprotein containing either two major protein species (or deficient in the lower molecular weight protein species). Polynucleotide phosphorylase eliminates the reverse transcription template activity of the lens mRNA. This effect is blocked in the messenger ribonucleoprotein. Such observations suggest that at least one of the protein components associated with lens messenger ribonucleoprotein may be located in the 3'-terminal region. Only a small variation in translation activity was observed between the messenger ribonucleoproteins and their respective mRNAs.