Human a,-macroglobulin is a tetrameric protein of M, 725 000 that interacts with proteases and primary amines. The conformational changes associated with a,-macroglobulin interaction with the natural substrate thrombin, other proteases, and methylamine were studied by using circular dichroism (205-350 nm), difference absorption spectroscopy (240-350 nm), and stopped-flow fluorescence spectroscopy. The far-ultraviolet circular dichroic spectrum of native a2-macroglobulin was characterized by a negative band at 215 nm, with shoulders at 210 and 221 f 1 nm, and is consistent with the presence of considerable ordered secondary structure. Reaction of the protein with proteases and methylamine leads to a small reduction in ellipticity between 205 and 230 nm. The absorption difference spectra of proteaseand methylamine-reacted a,-macroglobulin are similar in many respects, but some significant differences were noted. Protease-modified a,-macroglobulin exhibited major difference extrema at 282.5 and 293 nm, assigned to tryptophan, with shoulders at 289.5 and 296 nm. The position and magnitude of the 293-nm difference band are consistent with the perturbation of about 5.4 f 1.6 tryptophanyl residues in thrombin-reacted a2-macroglobulin. The nucleophile-converted protein is missing the 293-nm difference peak, and the 296-nm component is well resolved. The 289.5-and 296-nm bands are unusual, and firm assignments cannot be made. The
A B S T R A C T Two 0-hydroxylysyl glycosides, HylGal-Glc and Hyl-Gal, have been isolated from normal human urine and shown to be identical to two glycosides isolated from alkaline hydrolysates of collagen. A relatively sample and reproducible analytical procedure has been devised to measure the levels of these glycosides in human urine. By the use of this procedure it was shown that a normal diet has only a small effect on 24-hr urinary excretion levels of these glycosides indicating an endogenous origin. Urinary glycoside levels appear to be highest in children, roughly paralleling collagen turnover as indicated by urinary hydroxyproline levels. Collagen turnover equivalents calculated from urinary hydroxylysyl glycoside levels were found to be significantly larger than collagen turnover equivalents calculated from urinary hydroxyproline levels. This suggests that urinary glycosides are more quantitative indicators of collagen metabolism than urinary hydroxyproline.The ratio of Hyl-Gal-Glc to Hyl-Gal was measured sults it is postulated that the bimodal distribution of glycoside ratios represents two populations of collagen turnover, the lower ratio population having a high bone collagen turnover relative to the second population. Examination of the types of subjects making up the two populations supports this hypothesis. These data suggest that urinary 0-hydroxylysyl glycoside excretion, in addition to providing a more quantitative estimate of collagen turnover than urinary hydroxyproline, may prove to be of value as a specific means of studying the metabolism of bone collagen. INTRODUCTIONThe urinary excretion of hydroxyproline peptides is a commonly used standard for the evaluation of collagen metabolism (1). Elevated levels of excretion over normal have been observed in hyperthyroidism (2), acromegaly (3), burned patients (4), and patients with bone-destroying lesions such as Paget's disease of bone (3), rickets of vitamin D-deficiency (5), metastatic carcinoma (6), and extensive fractures (7). There has been general agreement that in each of these disorders elevated urinary hydroxyproline excretion is a reflection of an abnormally high rate of collagen metabolism.There are, however, at least two limitations to the use of urinary hydroxyproline peptide excretion as a measure of collagen metabolism. It has been estimated that less than 15% of the hydroxyproline of degraded collagen appears in the urine (8) so that urinary hydroxyproline peptides are poor indicators of total collagen
The rate of inhibition of creatine phos-this protection. This observation has allowed a more phokinase by iodoacetamide or p-nitrophenylacetate direct evaluation of previously reported substrateis reduced in the presence of an equilibrium mixture of dependent conformational changes in this enzyme, the substrates of the enzyme: MgATP2-, creatine, Studies of sedimentation constant, reduced viscosity, MgADP-, and phosphocreatine. It has now been shown deuterium exchange, and trypsin susceptibility indicate that the addition of the nonfunctional substrate pair, only small structural alternations in the protein can MgADP-and creatine, is necessary and sufficient for be related to the binding of the combined substrates.Q K_/amuels et al. ( 1961) have reported that creatine phosphokinase (CPK1) undergoes a substrate-dependent change in optical rotation which was interpreted as reflecting a conformational change in the protien. This change was reported to be dependent upon the simultaneous presence of both substrates, and it was suggested that it might play an important role in the mechanism of enzyme action. This hypothesis of a special configuration for "working enzyme" has been employed by Watts and Rabin (1962) to explain the protection against carboxymethylation with iodoacetamide of the two reactive sulfhydryl groups of the enzyme which is afforded by the substrate mixture.
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