Two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii, designated as DRTase I and DRTase II, were separated and purified almost to homogeneity by one-step affinity chromatography. DRTase I is distinguished by specifically catalyzing the direct transfer of 2-deoxyribosyl residues from purine deoxyribonucleosides to free purine bases, whereas DRTase II has a rather broad substrate specificity and is able to transfer the deoxyribosyl moiety between pyrimidines and between purines and pyrimidines. Furthermore, in addition to the different substrate spectrum, we clearly differentiated the two enzymes by comparing their varying temperature/activity and pH/activity profiles, their kinetic constants, their behaviour in Western blot analysis, and their N-terminal amino acid sequences. Denaturing and non-denaturing DISK-PAGE revealed strong evidence that both intact enzymes consist of hexamers with subunit molecular weights of approximately 20,000 for DRTase I and 18,000 for DRTase II.

Microvesicles (MVs) in endocrine cells are morphologically similar to neuronal synaptic vesicles. MVs were shown to contain proteins involved in neurotransmitter storage such as vacuolar H(+)-ATPase and neurotransmitter transporters, and ones in vesicular trafficking such as synaptobrevins and N-ethylmaleimide-sensitive fusion protein. Isolated MVs accumulate cell-specific neurotransmitters in an energy-dependent manner. Upon stimulation, the MVs may fuse with the plasma membrane and secrete the internal neurotransmitters. Thus, endocrine cells possess an MV-mediated secretion system as an intercellular signal transducing system.

The serpins illustrate the way in which the study of a protein family as a whole can clarify the functions of its individual members. Although the individual serpins have become remarkably diversified by evolution they all share a common structural pathology. We have previously shown how plotting of the dysfunctional natural mutations of the serpins on a template structure defines the domains controlling the mobility of the reactive centre loop of the molecule. Here we compare these natural mutations with reciprocal mutations in recombinants that restore the inhibitory stability of a labile member of the family, plasminogen activator inhibitor-1 (PAI-1). The combined results emphasise the critical part played by residues involved in the sliding movement that opens the A-sheet to allow reactive loop insertion. It is concluded that changes in these residues provide the prime explanation for the ready conversion of PAI-1 to the inactive latent state. The consistency of the overall results gives confidence in predicting the likely consequences of mutations in individual serpins. In particular the two common polymorphic mutations present in human angiotensinogen are likely to affect molecular stability and hence may be contributory factors to the observed association with vascular disease.

The influence of DNA methylation in vitro on the activity of the very strong human cytomegalovirus (HCMV) major immediate early (IE) modulator/enhancer/promoter region was investigated by transient transfection experiments of premonocytic HL-60 cells. While sequence-specific methylation of the major IE enhancer and/or modulator with the cytosine methyl-transferases FnuDII, HhaI and HaeIII had no significant effect, the promoter activity was completely repressed by methylation of the cytosine in 5'-CpG sites with the Spiroplasma methyltransferase SssI. Addition of TNF-alpha or PMA which are strong stimulators of HCMV major IE enhancer/promoter activity in premonocytic HL-60 cells had no effect on repression. Inactivation of the IE enhancer/promoter via methylation by M.SssI could be partially alleviated by co-transfection with an excess of untranscribable highly methylated DNA. These results indicate that a methyl-CpG binding factor is involved as mediator in the inhibitory effect of HCMV enhancer/promoter methylation. Taken together, the HCMV major IE enhancer/ promoter has been shown to be susceptible to transcriptional inactivation by methylation of the cytosines in CpG dinucleotides, a process that is proposed to play a modulatory role in viral latency.

The trifluoroethanol (TFE)-induced formation of alpha-helical structures in the peptide hormone calcitonin (salmon) was studied using limited proteolysis combined with capillary zone electrophoresis. A low TFE content in TFE/buffer mixtures was insufficient to introduce secondary structure, but two Lys-Leu bonds were found to have become inaccessible to proteolysis with clostripain. The influence of increasing helical degree of the Thr6-Lys18 (or Thr6-Tyr22 in the trans conformation) segment on the cis/trans isomerization of the adjacent Tyr22-Pro23 peptide bond was examined by means of isomer specific proteolysis. Results indicate that the helix dipole does not influence the cis/trans equilibrium distribution of the flanking Tyr22-Pro23 bond but considerably increases its isomerization rate Ko-->t.