Effects of age, sex, cataract, and cataract surgery on serum gamma-crystallin concentration.

PURPOSE: To determine if measurement of lens protein in serum is a feasible means to gain information on the physiologic status of the lens in human subjects. METHODS: The gamma-crystallin concentration was measured by a sandwich radioimmunoassay in the sera of 280 subjects aged 25-94 years. Medical records were reviewed for diagnoses of cataract and aphakia. RESULTS: There was no effect of age or sex on the serum gamma-crystallin concentration. There were 57 subjects with cataract and 27 with aphakia. gamma-Crystallin was higher in all cataract groups and lower in aphakia. The mean gamma-crystallin concentrations for selected subject groups were as follows: clear lens 301 pg/ml; pure nuclear cataract 344 pg/ml; pure cortical cataract 439 pg/ml and aphakia 255 pg/ml. CONCLUSIONS: This is the first published report to show that lens protein is measurable in serum and to demonstrate the feasibility of using serum assays of lens proteins to gain information on the physiological status of the lens. Our results confirm the hypothesis that molecular and cellular events leading to cataract cause increased leakiness of lens cell membranes with release of lens proteins appearing in the blood. It is conceivable that measurement of lens proteins in serum might find future use in the evaluation of cataract risk, potentially cataractogenic and anticataractogenic agents, retained lens fragments after phacoemulsification, secondary cataract, phacolytic glaucoma, anaphylactic endophthalmitis, eye injuries, and other eye diseases.

Tyrosine phosphorylation of phosphatase inhibitor 2.

Inhibitor 2 is a heat-stable protein that complexes with the catalytic subunit of type-1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosphorylated on tyrosine residues. Inhibitor 2 was 32P-labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type-1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type-1 phosphatase activity.

Tyrosine-phosphorylated calmodulin has reduced biological activity.

Calmodulin is phosphorylated by the purified insulin receptor on tyrosine residues with a maximum stoichiometry of 1 mol phosphate/mol of calmodulin. Isolated tryptic phosphopeptides were sequenced by manual Edman degradation and demonstrated that calmodulin is equally phosphorylated on tyrosine 99 and tyrosine 138. Phosphorylated calmodulin has a decreased affinity (K0.5 = 4.2 nM) for the 63-kDa isozyme of cyclic nucleotide phosphodiesterase compared to nonphosphorylated calmodulin (K0.5 = 2.1 nM). The K0.5 for Ca2+ is marginally increased from 2.8 to 3.2 microM in the presence of phosphotyrosyl calmodulin. The effect of the calmodulin antagonist, mastoparan, was investigated to determine whether mastoparan would differentially inhibit calmodulin- or phosphocalmodulin-dependent enzyme activity. The IC50 of mastoparan is fourfold lower for phosphotyrosyl calmodulin compared to nonphosphorylated calmodulin. Phosphorylation of calmodulin may provide a mechanism for the differential regulation of calmodulin-dependent enzymes. These observations further support a potentially important regulatory function of calmodulin phosphorylation in signal transduction.

The preparation of tritiated tunicamycin.

A relatively simple and inexpensive procedure was devised for the radiolabeling of the glycoprotein biosynthesis inhibitor, tunicamycin. The procedure is based on hydrogen exchange in alkaline solutions of tritiated water. It was noted that the antibiotic was much more alkali labile than model compounds such as uridine. The alkali stability of the inhibitor was studied to determine conditions for optimum labeling and yield. The effects of alkaline incubation on the inhibitory properties of the antibiotic were also investigated and it was found that the breakdown products are not effective inhibitors of the reaction that transfers N-acetylglucosamine-1-phosphate to dolichyl phosphate. The isolated radioactive tunicamycin homologs, however, retained all their inhibitory action. Incubation of tunicamycin in the presence of deuterated water and mass spectral analysis showed that under the conditions used for the tritiation of tunicamycin the major product exchanged six hydrogen atoms. The position of the tritium atoms in labeled tunicamycin was not determined. The radioactive label in these compounds was shown to be stable under physiological conditions and should be useful for investigations involving the action of these antibiotics.