Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in gamma-carboxyglutamic acid.

We have determined that the organic matrix of calcium oxalate kidney stones contains a glycoprotein inhibitor of calcium oxalate crystal growth (nephrocalcin) that resembles nephrocalcin present in the urine of patients with calcium oxalate stones and differs from nephrocalcin from the urine of normal people. Pulverized calcium oxalate renal stones were extracted with 0.05 M EDTA, pH 8.0; nephrocalcin eluted in five peaks using DEAE-cellulose column chromatography, and each peak was further resolved by Sephacryl S-200 column chromatography. Four of the five DEAE peaks corresponded to those usually found in nephrocalcin from urine; the fifth eluted at a lower ionic strength than any found in urine. Amino acid compositions and surface properties of nephrocalcins isolated from kidney stones closely resembled those of nephrocalcins isolated from urine of stone-forming patients: they differed from normal in lacking gamma-carboxyglutamic acid residues, and in forming air-water interfacial films that were less stable than those formed by nephrocalcin from normal urine.

Urine glycoprotein crystal growth inhibitors. Evidence for a molecular abnormality in calcium oxalate nephrolithiasis.

One reason that some people are prone to calcium oxalate nephrolithiasis is that they produce urine that is subnormal in its ability to inhibit the growth of calcium oxalate crystals. We have identified in human urine a glycoprotein (GCI) that inhibits calcium oxalate crystal growth strongly, and at low concentrations (10(-7) M); in this study, we have isolated GCI molecules from the urine of normal people and patients with calcium oxalate stones. GCI from stone formers is abnormal in three ways: it contains no detectable gamma-carboxyglutamic acid (Gla), whereas normal GCI contains 2-3 residues of Gla per mole; about half of the GCI in urine of patients inhibits crystal growth 4-20 times less than normal GCI as judged by its performance in a kinetic growth assay, in vitro; at the air-water interface, patient GCI has a film collapse pressure approximately half of normal. GCI molecules from the urine of patients with calcium oxalate nephrolithiasis are intrinsically abnormal, and these abnormalities could play a role in the genesis of stones.

The kinetics of activation of normal and gamma-carboxyglutamic acid-deficient prothrombins.

The kinetics of activation of normal and gamma-carboxyglutamic acid (Gla)-deficient prothrombins isolated from cattle maintained for extended periods on the vitamin K antagonist dicoumarol were studied. The catalyst was prothrombinase, comprising isolated Factor Xa, Factor Va, phospholipid vesicles, and calcium ion. The Km and kcat values for prothrombins with 0, 1, 2, 5, 7, and 10 Gla residues were determined both by initial rate analysis and by integrated Michaelis-Menten-Henri analysis. Each of the Gla-deficient prothrombins exhibited kcat values similar to that of normal 10-Gla prothrombin but Km values that were 8- to 20-fold greater than that of the normal molecule. The increased Km coincided with a loss of Ca2+- and phospholipid-binding properties of the Gla-deficient prothrombins. The magnitude of the defect in both the kinetics of activation and Ca2+ and phospholipid binding is not progressive with the loss of Gla residues but rather appears abruptly with the loss of as few as 3 of the 10 Gla residues present in the normal substrate. The theoretical relationship between Km(app) and the dissociation constant (Kd) of the prothrombin-phospholipid interactions was derived. According to the result, the increase in apparent Km observed with the Gla-deficient prothrombins corresponds to at least a 100- to 1000-fold decrease in affinity for phospholipid compared to the affinity of normal prothrombin. In addition, the products of the activation of 10-Gla prothrombin were found to inhibit the activation of the Gla-deficient prothrombins.