Insulin acidification

Renstrom, E., Ivarsson, R. and Shears, S.B., 2002, Inositol 3,4,5,6-tetrakisphosphate inhibits insulin granule acidification and fusogenic potential. J. Biol. Chem. 277 26717-26720. 

The glucagon/insulin ratio can rise under certain pathological conditions (i.e., insulin-dependent diabetes). A small percentage of diabetics develop ketoacidosis, a condition that results from the overproduction and underuhlization of ketone bodies. Increased concentrations of p hydmxybutyrate and acetoacetate, which are acids, can cause a drop in the pH of the blood. This acidification, known as acidosis, can impair the ablLity of the heart to contract and result in a loss of consciousness and coma, which, in rare cases, may be fatal. Diabetic ketoacidosis may manifest as abdominal pain, nausea, and vomiting. A subject may hyperventilate (breathe quickly and deeply) to correct acidosis, as described under Sodium, Potassium, and Water in Chapter 10. It is the responsibility of the clinician, when confronted with a subject whose breath smells of acetone or who is hyperventilating, to facilitate prompt treatment. 

Murphy, R. F. Powers, S. Cantor, C. R. Endosome pH measured in single ceUs by dual fluorescence flow cytometry rapid acidification of insulin to pH 6. J. CeU Biol. 1984, 98, 1757-1762. 

Secretin, an intestinal hormone obtainable from the duodenum, particularly on acidification, has been used clinically as a diagnostic aid in pancreatic disease to stimulate pancreatic secretion. It has been well-established that secretin also inhibits gastric acid secretion. The most recent investigation showed that secretin Inhibited gastrin-stimulated secretion but not that induced by histamine in dogs d.th a Heidenhain pouch.Insulin reD ase by the pancreas is stimulated by secretin in dogs 9 and in man. ... 

A difference spectrum involving a blue shift upon acidification and enzymatic digestion has been observed for insulin and ribonuclease, a typical difference spectrum being shown for insulin in Fig. 130. The main... 

Where difference spectra are obtained from proteins by a change of pH or solvent, or by enzymatic digestion there is no a -priori basis for assigning their prigin to any one or more of the above effects. Nor is it possible at present to predict the relative magnitude of the various effects. However, for proteins such as insulin and ribonuclease where the amino acid sequences are known, and the difference spectra have been examined in more detail, it should be possible to exclude some of the possible effects. In addition, for insulin, ribonuclease, and other proteins, the shift upon denaturation, acidification, or proteolysis is always toward shorter wave-lengths, so we may conclude that those solvent effects which lead to a red shift do not preponderate in the over-all spectral change. 

The same type of difference spectrum, as shown in Fig. 130, is obtained if native insulin at pH 1.6 is measured against native insulin at pH 7.9 (Fig. 134). Presumably a tyrosyl hydrogen bond in the native molecule is ruptured by putting a proton on the acceptor group. If the same acidification experiment is carried out with the tryptic digest, a similar difference... 

Fig. 134. Comparison of the difference spectra obtained by tryptic digestion, by acidification of native zinc-insulin, and by acidification of the trypsin digest of zinc-insulin. Optical density differences were measured in 0.5-cm. cells using approximately 0.5% solutions of insulin. In the figure, all readings are adjusted to 1-om. cells for 0.5% insulin (Laskowski e< al., 1960a).

The very same principle was applied by Suzuki et al. [19] for the purification of the placental insulin receptor, starting with a prepurified sample, obtained from a plasma membrane fraction of human placenta by a fractionation protocol including affinity chromatography on Sepharose-concanavalin A as the last step. The following purification step was carried out, using insulin bound to dextran (M 40000) as the biospecific, water-soluble carrier. After filtration of the dextran-insulin-receptor complex on an adequate gel and then dissociation by mild acidification, the insulin receptor was obtained in a purified form. This method permitted a 8700 fold purification, from crude plasma membrane and, even more remarkable, a 60-fold purification after the affinity chromatography step. 

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