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Tissue-nonspecific alkaline phosphatase (TNAP) is a Zn metalloenzyme with two Zn ions bound to the M1 and M2 catalytic sites that functions to control the phosphate\/pyrophosphate ratio during biomineralization. Here, we studied aortas from Tagln-Cre <jats:sup>+\/\u2212<\/jats:sup>; Hprt<jats:sup>ALP\/Y<\/jats:sup> TNAP overexpressor (TNAP-OE) mice that develop severe calcification. Zn histochemistry was performed using the sulfide-silver staining method in combination with a Zn partial extraction procedure to localize mineral-bound (mineral Zn) and TNAP-bound Zn (tenacious Zn), since soluble Zn (loose Zn) is extracted during fixation of the specimens. Two synthetic bone mineral composites with different Zn content, bone ash, and rat epiphyseal growth plate cartilage were used as controls for Zn staining. In order to correlate the distribution of mineral and tenacious Zn with the presence of mineral deposits, the aortas were examined histologically in unstained and stained thin sections using various light microscopy techniques. Our results show that 14 and 30 dpn, TNAP is concentrated in the calcifying matrix and loses Zn as Ca<jats:sup>2+<\/jats:sup> progressively displaces Zn<jats:sup>2+<\/jats:sup> at the M1 and M2 metal sites. Thus, in addition to its catalytic role TNAP has an additional function at calcifying sites as a Ca-binding protein.<\/jats:p>\n                <jats:p><jats:bold>Graphical Abstract<\/jats:bold><\/jats:p>","DOI":"10.1007\/s10735-024-10207-3","type":"journal-article","created":{"date-parts":[[2024,6,8]],"date-time":"2024-06-08T05:01:47Z","timestamp":1717822907000},"page":"465-479","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Zinc-alkaline phosphatase at sites of aortic calcification"],"prefix":"10.1007","volume":"55","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3156-0989","authenticated-orcid":false,"given":"Santiago","family":"Gomez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1547-2671","authenticated-orcid":false,"given":"Jos\u00e9 Luis","family":"Mill\u00e1n","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,6,8]]},"reference":[{"issue":"6","key":"10207_CR1","doi-asserted-by":"publisher","first-page":"1711","DOI":"10.1016\/S0002-9440(10)62481-9","volume":"166","author":"HC Anderson","year":"2005","unstructured":"Anderson HC, Harmey D, Camacho NP, Garimella R, Sipe JB, Tague S, Bi X, Johnson K, Terkeltaub R, Mill\u00e1n JL (2005) Sustained osteomalacia of long bones despite major improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline phosphatase\/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice. 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