Spielman A I, Bernstein A, Hay D I, Blum M, Bennick A
Department of Biochemistry, University of Toronto, Ontario, Canada.
Arch Oral Biol. 1991;36(1):55-63. doi: 10.1016/0003-9969(91)90054-x.
Human saliva is supersaturated with respect to basic calcium phosphate salts but is stabilized by specific macromolecules that inhibit calcium phosphate precipitation. One of the families of inhibitory proteins in human and monkey saliva is the acidic proline-rich proteins. The purpose of this study was to isolate and characterize inhibitors of calcium phosphate precipitation from rabbit parotid saliva. Saliva was fractionated by immunoaffinity chromatography and anion exchange chromatography. Individual fractions were assayed for their ability to inhibit calcium phosphate crystal growth and the fraction associated with the inhibition was purified by repeated anion exchange chromatography, preparative gel electrophoresis and electroelution. A major (APRP) and two minor proteins (AM1, AM2) that were inhibitory were purified. APRP is an acidic proline-rich phospho-glycoprotein and a very potent inhibitor of secondary crystal growth of calcium phosphate as it was active at a concentration of 2 x 10(-8) M in a standard assay. The N-terminal sequence of one APRP was EYENLDGSLAATQNDDD?Q and a clostripain fragment of APRP had the following N-terminal sequence PQHRPPRPGGH-????SPPP?GN???PPP. Although the N-terminal segment of APRP does not resemble that of proline-rich proteins, alignment of the clostripain fragment with the repeat region of such proteins from rat, mouse, monkey and man revealed a high degree of similarity, indicating a structural relationship with the proline-rich protein family.
人类唾液相对于碱性磷酸钙盐是过饱和的，但通过抑制磷酸钙沉淀的特定大分子得以稳定。人类和猴唾液中的抑制性蛋白质家族之一是富含脯氨酸的酸性蛋白。本研究的目的是从兔腮腺唾液中分离并鉴定磷酸钙沉淀的抑制剂。通过免疫亲和色谱和阴离子交换色谱对唾液进行分级分离。检测各个级分抑制磷酸钙晶体生长的能力，通过重复阴离子交换色谱、制备性凝胶电泳和电洗脱对与抑制作用相关的级分进行纯化。纯化出一种主要的抑制性蛋白（APRP）以及两种次要蛋白（AM1、AM2）。APRP是一种富含脯氨酸的酸性磷酸糖蛋白，是磷酸钙二次晶体生长的非常有效的抑制剂，因为在标准测定中它在2×10⁻⁸ M的浓度下具有活性。一种APRP的N端序列为EYENLDGSLAATQNDDD?Q，APRP的梭菌蛋白酶片段具有以下N端序列PQHRPPRPGGH -????SPPP?GN???PPP。尽管APRP的N端片段与富含脯氨酸的蛋白质的N端片段不同，但该梭菌蛋白酶片段与大鼠、小鼠、猴和人的此类蛋白质的重复区域比对显示出高度相似性，表明与富含脯氨酸的蛋白质家族存在结构关系。
