Distribution of manganese in rat pancreas and identification of its primary binding protein as pro-carboxypeptidase B

Abstract

Distribution of manganese (Mn) and its binding to specific proteins were examined in rat pancreas. A MnCl2 solution was injected subcutaneously into Wistar rats daily at a single dose of 15 mg of Mn/kg body weight for 10 days and the animals were killed 1 day after the last injection. The concentration of Mn in the pancreas increased considerably from 1.4 +/- 0.2 (control) to 13.3 +/- 3.7 micrograms/g wet tissue by the repeated injection of Mn. The distribution of Mn in the soluble fraction of the pancreas (170,000 g supernatant) was determined on a gel-filtration column (Asahipak GST-520) using an h.p.l.c.-inductively coupled argon plasma atomic-emission spectrometry (i.c.p.) technique. The metal was eluted as a single peak in the high-molecular-mass protein fraction, where Mn had been observed as a small peak in the control profile, suggesting that the administered Mn was bound to the same Mn-binding component as that in the control. On the basis of enzymic and chemical characterization of the protein, it was identified as a zymogen of carboxypeptidase B (pro-carboxypeptidase B, pro-CPB). The elution profiles of the protein by h.p.l.c.-i.c.p. indicated that Mn and zinc (Zn) were bound to the zymogen with a molar ratio of 1:4 in normal rat pancreas. Mn bound to the zymogen was easily replaced by Zn in vitro, suggesting that Mn was bound to the Zn-binding site and that the binding affinity to Zn was higher than that to Mn. The present results indicate that pro-CPB is the primary Mn-binding protein in the pancreas of control and also Mn-administered rats.

β‐Cell subcellular localization of glucose‐stimulated Mn uptake by X‐ray fluorescence microscopy: implications for pancreatic MRI

“…Lacking a suitable staining technique for Mn, most studies only assess Mn amounts in cells and tissues using atomic absorption spectroscopy or inductively coupled plasma–mass spectroscopy (ICP‐MS) , or by means of radioactivity using 54 Mn . All techniques are fairly accurate and have appropriate detection limits.…”

“…Two studies on isolated rodent pancreatic islets used Mn to study Ca handling in β-cells and found a significant Mn uptake following glucose stimulation in all fractions, except for microsomes, with the mitochondrial fraction having the highest percentage increase (30,31). Lacking a suitable staining technique for Mn, most studies only assess Mn amounts in cells and tissues using atomic absorption spectroscopy (15,(30)(31)(32)(33) or inductively coupled plasma-mass spectroscopy (ICP-MS) (16,28), or by means of radioactivity using 54 Mn (19,22,34). All techniques are fairly accurate and have appropriate detection limits.…”

“…Nonetheless, only a handful of studies so far have investigated distribution of the element in the organ with no distinction between exocrine and endocrine components. Mn was found to accumulate primarily in lysosomes where it is bound to pro‐carboxypeptidase B, a zymogen found in the exocrine portion of the pancreas . Two studies on isolated rodent pancreatic islets used Mn to study Ca handling in β ‐cells and found a significant Mn uptake following glucose stimulation in all fractions, except for microsomes, with the mitochondrial fraction having the highest percentage increase .…”

Manganese metabolism in humans

“…They found that Mn concentration in the pancreas was significantly increased from 1.4. to 13.3. mg/g wet tissue, and Mn was found in the high-molecular-mass protein fraction (66). Interestingly, they identified that Mn bound to the Zn binding site of protein zymogen of carboxypeptidase B (pro-CBP), which was the primary Mn-binding protein in the pancreas (66). In pancreatic islets isolated from ob/ob mice incubated with 0.2.5 mM Mn for 1 hour, the intracellular Mn concentration was about 25-fold higher than that of the extracellular medium which was stimulated by 20 mM D-glucose due to inhibition of Mn efflux in the islets (67).…”

“…Kodama and colleagues studied Mn distribution in Wistar rats by subcutaneous delivery of 15 mg of Mn/kg daily for 10 days. They found that Mn concentration in the pancreas was significantly increased from 1.4. to 13.3. mg/g wet tissue, and Mn was found in the high-molecular-mass protein fraction (66). Interestingly, they identified that Mn bound to the Zn binding site of protein zymogen of carboxypeptidase B (pro-CBP), which was the primary Mn-binding protein in the pancreas (66).…”