However, when I was asked to write about a new assay for semicarbazide-sensitive amine oxidases (SSAOs), my enthusiasm waned. This is a subject about which I know nothing, so I searched the literature to learn as much as I could. After reading several review articles I was able to write this scintillating paragraph:
Amine oxidases can be divided into two basic classes: Monoamine Oxidases (MAOs), which are flavin-containing enzymes with primary, secondary and tertiary amine groups as their substrates, and the Semincarbazide-Sensitive Amine Oxidases (SSAOs), which are copper-containing amine oxidases that deaminate primary amine groups to their corresponding aldehyde and produce ammonia and hydrogen peroxide.
Actually, the literature search did help, at least now I had a conceptual framework of where SSAOs fit in the big picture, right next to MAOs. Unfortunately I kept reading and learned that they catalyze their reaction via a Schiff base mechanism. That tidbit brought back a memory of a particularly unpleasant biochemistry exam in graduate school that gave me the shudders.
To recover, I went for a long walk, something else that I do when my writing isn’t going very well. I have been known to compose entire pieces while walking. On my walk I saw two Sandhill Cranes, who stared at me, unsure whether or not this science writer who was walking and mumbling about semicarbazides was a threat. I decided to do another literature search when I returned to my desk to see if Sandhill Cranes had any interesting, documented SSAO defects—something on which I could hang the rest of my writing. They don’t.
But SSAOs Are Really Cool…
It turns out though that SSAOs are really important enzymes. They exist in tissue and plasma forms, and elevated SSAO plasma levels are associated with congestive heart failure, Type I and Type II diabetes, Alzheimer disease and inflammatory diseases. Furthermore, SSAO activity in serum shows a positive correlation with body mass index (BMI). This class of enzymes functions in many critical processes such as glucose homeostasis and lymphocyte adhesion.
SSAOs are expressed in many tissues including smooth muscle layers of blood vessels, endothelial cells. Adipocytes, chondrocytes, fibroblasts, retina, sclera, kidney, spleen, placenta, umbilical artery and bone marrow tissues also exhibit SSAO activity.
The most important endogenous substrates for SSAOs in humans appear to be methylamine and aminoacetone. Methylamine is derived from epinephrine, creatine and creatinine metabolism, and it can be ingested or inhaled from food, drink and cigarette smoke. Bioactive amines, such as tyramine, histamine and dopamine, are also substrates for SSAO, but in physiological systems, MAOs appear to be the major enzyme involved in metabolizing these substances.
When SSAOs catalyze the deamination of methylamine, formaldehyde is produced, and formaldehyde is a highly toxic carcinogen. Methylgloxal one of the products of their activity on methylamine is also toxic, able to cross link proteins. Hydrogen peroxide, also a product of SSAO-catalyzed reactions, is a reactive oxygen species (ROS), and has been implicated in neural toxicity and other pathologies.
And a Rapid Screening Assay is Really Important
Given all of the physiological processes in which SSAO is involved, and the association of increased serum activity of SSAOs with many diseases, SSAOs are potentially interesting drug targets. And, they are also important for profiling compounds active against other enzymes to look for off-target effects.
Up to this point, the methods to study SSAO activity involve laborious techniques like HPLC, or expensive radioisotopes that present disposal issues, so an assay that is rapid, inexpensive and amenable to high-throughput applications is highly desirable. Peet and colleagues have recently reported the adaptation of the luminescent MAO-Glo™ Assay to screen for SSAO activity in 96- and 384-well formats. The assay was tested with different SSAO enzymes from both recombinant and cellular sources, and the authors demonstrated its utility in initial HTS activities, counterscreening and cellular profiling. They were able to demonstrate detection of known VAP-1 inhibitors in a high-throughput screen of nearly 900,000 compounds, using recombinant enzyme with statistical indicators showing the assay to be highly robust.
This modified assay could be a real boost to research that seeks to identify specific SSAO inhibitors, help identify off-target effects of drug compounds on SSAOs and generally increase our understanding of how these enzymes influence so much physiology.
So, yes when presented with the topic of a new screen for SSAOs, initially I wasn’t all that enthusiastic, but in the process I learned a lot of biology about an interesting class of enzymes. I used my degree, stimulated my brain, and well, enjoyed myself. It’s why I do what I do.
- Peet, G.W. et al. (2011) Bioluminescent method for assaying multiple semicarbazide-sensitive amine oxidase family members in Both 96- and 384-well plates. J Biomol. Scr. 16, 1106–11.
- Magyar, K. et al. (2001) Semicarbazide-sensitive amine oxidase. It’s physiological significance. Pure Appl. Chem. 73, 1393–1400.
- Yu, P.H. et al. (2003) Physiological and pathological implications of semicarbazide-sensitive amine oxidase. Biochim. Biophys. Acta. 1647, 193–9.
- Valley, M.P. et al. (2006) A bioluminescent assay for monoamine oxidase activity. Anal. Biochem. 359, 238–46.