Peter Bohley

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii.

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green algaChlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtiicultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors ofS-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.

The Fates of Proteins in Cells

Nascent polypeptide chains are in a dangerous situation as soon as they leave their place of birth, the channel of the large ribosomal subunit: more than 20 different pathways for the degradation of proteins exist in cells. Chaperones protect and guide the young protein molecules and support their correct foldings. Targeting signals direct the proteins to the organelles of their destination. The lysosome is the site of random degradation, while the proteasome is highly selective. Although these two organelles provide the most important pathways for the degradation of long- and short-lived proteins, other pathways with roles in deciding the fate of cellular proteins must also be considered.

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Arginylation, surface hydrophobicity and degradation of cytosol proteins from rat hepatocytes.

Intracellular proteolysis is necessary to adapt the protein composition of cells to environmental changes, to correct errors in transcription or in traslation, to degrade inactive pre- and pro-peptides and to liberate amino acids as an energy source from superfluous protein molecules in case of need1.

Lysosomal Proteolysis in Cultured Hepatocytes

We have investigated the degradation of labelled rat-liver cytosol proteins after their introduction (‘micro-injection’) into cultured hepatocytes by published procedures. These entailed incubation in a sucrose-PEG* medium, then incubations in a hypotonic medium, thereby facilitating selective lysis of the pinosomes without increasing the cytosolic activities of lysosomal enzymes. These treatments were without detriment to the survival rates of the hepatocytes in subsequent monolayer culture, during which the distribution and degradation of the introduced proteins was investigated by microscale cell fractionation and determination of protein-bound and free radioactivity in the subcellular fractions. Unexpectedly, lysosomes showed the highest selectivity for the uptake and degradation not only of long-lived but also of very short-lived cytosol proteins. Proteolysis was also investigated with isolated fractions.