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An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam). All sequences available on-line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography. While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms. Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms. Other helices which seem to exist in all P-450 forms include helices C, D, G and J. K. In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences.

For many studies on matrix metalloproteinases in immunohistochemistry it is important to be able to distinguish between the zymogen and activated forms of the enzymes. Activated human matrix metalloproteinase-9 (MMP-9, gelatinase B) was produced from the proenzyme by limited digestion with trypsin. The products of cleavage were characterised by SDS/PAGE and N-terminal sequencing. Trypsin treatment led to a stepwise removal of the propeptide domain and also caused cleavage within the C-terminal domain. Monoclonal antibodies specific for the activated form of human MMP-9 were raised by using a peptide corresponding to the N-terminus of the activated enzyme as immunogen. The antibodies do not recognise the MMP-9 proenzyme or the active or proenzyme forms of matrix metalloproteinase-2 (MMP-2, gelatinase A) and do not react with unrelated proteins in an unfractionated tissue extract. The antibodies were used to detect, by immunohistochemistry, activated MMP-9 in formalin-fixed, wax-embedded sections from a series of oesophageal cancer cases previously shown to contain MMP-9. All of the tumours contained activated MMP-9 localised to tumour cells and macrophages. As the antibodies are effective in immunohistochemistry on formalin-fixed, wax-embedded sections, they should prove useful for the detection of activated MMP-9 in various disease processes.

Isolation of newly synthesised RNA can be achieved by treatment of cells in culture with 6-thioguanosine or 4-thiouridine followed by separation of thiol-containing RNA by affinity chromatography on mercurated cellulose columns. After short periods of treatment with 6-thioguanosine the proportion of RNA retained on mercurated cellulose is the same for both poly(A)-containing and poly(A)-free RNA, indicating similar incorporation of the drug into mRNA and rRNA. However, after longer periods of exposure, the cytotoxic effect of 6-thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein; this suggests that synthesis of both RNA and protein are impaired. On the other hand, even after long exposure to high concentrations of 4-thiouridine, the syntheses of RNA and protein are not significantly affected. Proteins synthesised after treatment of cells with 6-thioguanosine are less stable than proteins synthesised after treatment of cells with 4-thiouridine.