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Methylglyoxal bis(guanylhydrazone) (MGBG) was incorporated extensively into baby hamster kidney (BHK-21/C13) cells by a temperature - and Na⁺, K⁺ - ATPase linked mechanism. The uptake of the drug was dependent on the growth state of the cells and transformed cells (PyY-cells) incorporated MGBG to a greater extent than normal BHK cells. The uptake of the drug was inhibited by coadministration with spermidine, spermine and N¹-acetylspermine and to a lesser extent by putrescine and Mg²⁺ ions. MGBG and spermine were cytotoxic and both compounds stimulated spermidine acetyltransferase (SAT) activity in BHK cells but not in PyY-cells suggesting that transformation enhanced resistance of the cells.SAT activity was present in both the nuclear and cytosolic compartment of both cell-lines and MGBG stimulation resulted in an increase in both N¹- and N⁸-acetylspermidine production in the nucleus and almost exclusive N¹-acetylspermidine production in the cytosol. MGBG stimulated spermine acetyltransferase activity in both nuclear and cytosolic compartments of mammalian cells. Increased intracellular acetylation after MGBG-treatment resulted in enhanced excretion of putrescine and N¹-acetylspermidine from both BHK- and PyY-cells implicating acetylation as a means to control intracellular polyamine content. MGBG was itself acetylated in the nucleus of both cell-lines, as was putrescine. However, prior MGBG treatment inhibited the acetylation of MGBG itself and the diamine suggesting that the two compounds are acetylated by a common nuclear acetyltransferase.A cytosolic spermidine acetyltransferase enzyme was purified over 2000-fold from BHK-cells and was found to acetylate putrescine, spermidine, spermine and MGBG. The activity of the enzyme preparation to all these compounds suggests that the enzyme preparation contained nuclear acetyltransferase activity. A model is preposed for the role of acetylation in both the nuclear and cytosolic compartments of mammalian cells.

The mechanism of protein sorting to the vacuole in yeast was studied both in vitro and in vivo. A series of experiments were performed to reconstitute transport of carboxypeptidase Y (CPY) from Golgi vesicles to vacuoles. In order to investigate this process, microsomes were purified from sec, pep4-3 mutant strains that accumulate inactive proCPY in the Golgi when incubated at the nonpermissive temperature. These were mixed with purified vacuoles isolated from a mutant lacking CPY activity, but containing active proteinases A and B. Transported proCPY is maturated by these proteinases to active form. Experiments indicate that maturation of CPY is due to the correct transport of proCPY from microsomes to vacuoles because:- Firstly, the reaction is temperature sensitive, requires ATP and is stimulated by the addition of soluble factors (S100). Secondly, the addition of proteinase A and B inhibitors to the reaction mixtures has a negligible effect on the maturation process. Thirdly, disrupting the membranes by the addition of Triton X-100 before addition of the proteinase inhibitors, inhibited the maturation of proCPY. Fourthly, the majority of CPY activity was observed in the sedimented fraction of the reaction mixtures rather than supernatant fractions. Lastly, analysis with western blot shows a clear band of mature CPY only in the sedimented fraction of the reaction mixtures with ATP. This in vitro system will be invaluable in investigating the molecular events of vacuolar biogenesis. For in vivo sorting of proteins to the vacuole, a series of experiments were performed that involved the genetic fusion of the CPY promoter and prepro-sequence of CPY to the bacterial Gus (β-glucuronidase) reporter gene. The Gus gene was expressed in yeast with high efficiency and the results of sub-cellular fractionation indicated that the Gus product was distributed in all cell components. Using a centromeric vector gave similar results but with a lower efficiency of Gus expression. Removal of 90bp from Gus, including Gus initiation codon does not completely inhibit Gus expression either in bacteria or in yeast. Fusion of the shortened Gus with the CPY prepro-fragment and expression in yeast led to the correct sorting of the CPY-Gus hybrid protein to the vacuole. This CPY-Gus fusion is potentially useful in the genetic analysis of mutations defective in vacuolar protein sorting.

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