Human urinary protein with high calcium affinity

75 Clinica Chimica Acta, 56 (1974) 75-81 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CCA 6612

HUMAN URINARY PROTEIN WITH HIGH CALCIUM AFFINITY

L. TEJLER, F. LINDGARDE, J. MALMQUIST and 0. ZETTERVALL Department of Medicine, University of Lund, Malmti General Hospital, S-21 4 01 Malmb’ (Sweden) (Received May 8,1974)

Summary Concentrated human urines have been investigated. By electrophoresisradioautography after 4 ’ Ca addition an electrophoretically homogeneous calcium ligand of (Y?mobility was found in all samples. The ligand is a protein with a molecular size corresponding to a molecular weight of approximately 42 000. The calcium binding affinity is very high since binding of calcium ions occurs even in the presence of high molar excess of Na2 EDTA. Urines from three patients with hypercalciuria contained larger than normal amounts of the calcium binding protein.

Introduction Calcium binding proteins have been isolated from small intestine in man and other species [l-3], as well as from bovine cortical bone [4,5], human kidney [6], porcine parathyroid [7] , and from the brains of squid and cuttlefish [ 81. The intestinal calcium binding protein seems to play an important role in calcium absorption [9] , while the functional roles of the other calcium binders are uncertain. In those cases where the association constant for calcium has been determined [8,9], the proteins have been found to have a considerably higher calcium affinity than human serum albumin [lo]. Albumin is believed to be the quantitatively predominant calcium binding intravascular protein. It can be characterized as a high capacity, low affinity calcium carrier, acting as a calcium ion buffer. It is not known whether calcium binding macromolecules are involved in renal calcium handling, and the influence of such molecules on urinary calculus formation has been debated for some years [ll]. With the purpose of detecting calcium binding serum proteins an electrophoresis-radioautography method has been developed [12]. The present report concerns the application of this method to human urinary concentrates.

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Material and Methods Urine samples

These were obtained from 8 healthy individuals and from 7 patients with the following conditions: hypercalciuria (urinary calcium exceeding 300 mg/24 h on controlled diet) and renal calculus formation, malignant reticulosis with hypercalcemia and hypercalciuria, hyperparathyroidism (surgically verified), chronic pyelonephritis with uremia, and lupus glomerulitis. Urine was collected in plastic bottles with sodium azide added as preservative. After each 24-h collection was complete the urine was frozen and stored at -20”. Concentration of urine This was done by pressure ultrafiltration, after passage through a paper filter. An Amicon Diaflo type 402 cell with a PM 10 membrane (nominal retention limit around mol. wt 10 000) was employed. The cell was pressurized with nitrogen (50 psi). In some cases, concentrates from pressure ultrafiltration were further concentrated by dialysis against polyethylene glycol (average mol. wt 20 000). Union Carbide 23/32 inch tubing was used. Both concentration procedures were carried out at 6-8’. After concentration the preparations were centrifuged at 15 000 X g for 30 min. The concentration factor varied between 300 and 1000. Protein concentration This was determined by the method of Lowry et al. [13] as modified by Hartree [14]. Human serum albumin was used as standard.

s

( 4 Cal Cl2

[45Ca]Clz, containing 20-60 1.18calcium per ml, spec. act. 0.88-1.0 mCi/ml, was purchased from Amersham. one-dime~~siona~ electr~phores~s with 4 5Ca and radioautograph~ These processes were performed as described previously [12] . Barbital buffer, 0.075 M, pH 8.6, without calcium addition, was used throughout. Concentrated urine samples recieved l/10 volume isotope solution before electrophoresis. In some experiments the urine concentrates were dialysed before 4 ’ Ca addition. Dialysis, in Union Carbide 8/32 inch tubing, was either against 0.023 M Tris-HCl-0.12 M NaCl, pH 7.5 (2 X 2500 volumes 72 h), or against 0.01 M Na, EDTA-0.023 M Tris-HC1-0.12 M NaCl, pH 7.5 (1250 volumes, 24 h), followed by Tris-NaCl without EDTA (2 X 2500 volumes, 48 h). Temperature at dialysis was 6-8”. Gel filtration Sephadex G-100 (Pharmacia) was used. The buffer was 0.025 M TrisHCi-0.12 M NaCl, pH 7.5. Column size was 25 mm X 385 mm. The separations were carried out at room temperature. The effluent was collected in 3 ml fractions at a flow rate of 7 ml/h. Cytochrome c, mol. wt 12 400 (Miles), bovine serum albumin, mol. wt 69 000 (Sigma), and ovalbumin, mol. wt

77

45 000 (Miles) were run separately as molecular weight markers. Gel filtration fractions were analysed for calcium binders by electrophoresis after 4 ’ Ca addition.

Proteolytic

digestion

Urine concentrates, 12 were incubated with trypsin 4 ’ Ca 3 h at 37”. Thereafter for subsequent electrophoresis. All solutions used were

mg protein per ml, in 0.046 M Tris-HCl, pH 8.1, (Difco) at a final concentration of 0.3 mg/ml, for was added and the incubate was left for 48 h at 4” prepared with twice deionized

water.

Results In all urines, a calcium binding fraction was visible on the radioautogram in the cr2 region. In some of the urine concentrates, a corresponding band was clearly seen on the protein stained electrophoresis plate. However, in other concentrates there was no unequivocal protein band corresponding to the radioautogram. The processing of 0.15 M NaCl through the various concentration steps described followed by electrophoresis-radioautography after 4 ’ Ca addition did not result in any bands either on the protein stained electrophoresis plate or on the radioautogram. Accordingly, there was no indication that any calcium binding material was extracted from any part of the equipment used. In urine from one of the patients (case J.K., recurrent stone formation

NS I

JK

JK II

NU

JK NU -diatysed-

Fig. 1. Agarose gel electrophoresis. I: protein stain. Normal serum (NS), urine concentrate (JK), (protein content 15.5 mglml) from case J.K. (idiopathic hypercalciuria) and normal urine concentrate (NW) (protein content 13.8 mglml). II: 45Ca-radioautogram. Dialysed: urine concentrates dialysed against NazEDTA prior to 45Ca addition.

with hypercalciuria) the calcium binding fraction was present in higher concentration than in normal urine (Fig. 1). The same finding was made in two additional cases of hypercalciuria. The uptake of 4 ’ Ca increased considerably after EDTA dialysis, as evidenced by increased intensity of the radioautogram band (Fig. 1). No other sharply delineated calcium binding fractions were visible on the radioautograms. However, after dialysis a weak radioactive band appeared in the fl region (Fig. 3). All of the concentrates contained albumin, but the present electrophoretic method does not visualize binding of calcium to albumin

[121*

Many electrophoretic protein patterns showed some degree of smearing, i.e., a background stain extending from the application slit towards the cy region. Calcium binding corresponding to this stain was evident on the radioautograms. The latter also showed some radioactivity located diffusely on the cathodal side of the slit. Some of this electrophoretically polydisperse material most probably represents Tamm-Horsfall glycoprotein. This material has been shown to bind calcium [15].

Characterization of the calcium binding urinary component Tryptic digestion of urinary concentrates caused a decrease

in both protein staining and calcium binding of the component, thus establishing that the calcium binder is a protein. On gel filtration on Sephadex G-100 the protein emerged between bovine serum albumin and cytochrome c, at a position almost coinciding with that of ovalbumin (mol. wt 45 000) (Fig. 2). The calcium binding protein was not visible as a discrete protein peak in the elution pattern, but was part of a large peak emerging after urinary albumin. The localization of the calcium binding urinary protein (CaBUP) was demonstrated by electrophoresis-radioautography of all fractions. In four of the fractions, calcium binding material was clearly visible (Fig. 2). Calculations based on V, /V, values [161 indicated a molecular size corresponding to a mol. wt of about 42 000.

EFFLUENT

WLLME

(ml)

Fig. 2. Fractionation on Sephadex G-100 of 2 ml normal urine concentrate (protein content 12.5 mg/ml). The arrows indicate the elution volumes for bovine serum albumin, ovalbumin and cytocbrome c, respectively. The histogram indicates the ^mount of calcium binder in fractions as estimated from radiautographic band intensity after 45Ca addition and electrophoresis.

79

NS

NU

A

E 1Omrn

C

A

B 2h

C

A

f3 6h

c

5% I

5 LB!?

c:

I

II

1

Fig, 3. Agarose gel electrophoresis. I: protein stain. Normal serum (NS) and normal (NU). II: 45&a-radioautogram. A, NU dialysed against Trk-NaCl without NqEDTA; against NazEDTA followed by Tris-NaCl; C, NU non-dialysed. Periods of incubation electrophoresis are indicated. Protein contents of all samples in I and II 14 m&ml.

urine concentrate B, NU dialysed with 45Ca before

Binding studies (Fig. 3). Binding of 4 ’ Ca to CaBUP was time-dependent. Maximal radioautographic visibility required incubation of urinary concentrate with isotope for at least 6 h. Dialysis of concentrates against Tris-NaCl before isotope addition caused a more rapid 4 ’ Ca uptake. This acceleration of calcium binding was even more pronounced when the preparations had been dialysed against EDTA before Tris-NaCl dialysis. Not only was the calcium uptake more rapid after either dialysis, but in addition the final amount of isotope taken up was markedly increased. On sim~~eous addition of 4 ’ Ca and a large (83-fold) molar excess of EDTA, isotope binding still occurred, although to a far lesser extent (Fig. 4). Similarly 4 ’ Ca bound to CaBUP could partially be removed by the subsequent addition of EDTA or by the addition of non-radioactive calcium but not by Tris-NaCl buffer. f

A

8

C

Fig. 4. Electrophoresis-radioautography of normal urine concentrate (protein content 20 mg/ml) pmdialysed against Tris-N&l. A: Urine concentrate plus l/4 volume Tris-N&l containing 250 nmoles EDTA prior to 45Ca addition (total calcium content 3 nmoles). B: Urine concentrate plus l/4 volume Tris-N&l. 45Ca addition as in A. C: NqEDTA and 4 %a as in A was first mixed before the addition of urine concentrate.

80

Discussion The experiments demonstrate that human urine contains a calcium binding electrophoretically homogeneous substance of (Y~ mobility. This substance is probably of protein nature as its calcium binding capacity and its uptake of the protein stain, Coomassie Brilliant Blue, both could be decreased by proteolytic digestion, That indeed the bound radioactivity represents 4 ’ Ca and not radioactive impurities was confirmed in competition experiments. Addition of non-radioactive calcium ions to protein-’ 5 Ca complex decreases the amount of protein bound radioactivity. The calcium ion affinity of this protein (CaBUP) is very high since appreciable binding could occur even in the presence of Naz EDTA in large molar excess over calcium ion and ligand. Whether CaBUP binds calcium ions in vivo is not known. However, prolonged dialysis, or treatment with Naz EDTA and subsequent dialysis, of concentrated urine increased the 4 ’ Ca binding capacity of the protein as well as the rate of 4 ’ Ca uptake. This suggests that the number of sites available for of previously bound calbinding of 4 ’ Ca had been increased by dissociation cium ions from the protein. That indeed EDTA treatment removes bound calcium ions was confirmed in another experiment. Hence, the protein as existing in concentrated urine probably contained bound calcium ions. The molcular size of the protein corresponds to an approximate mol. wt of 42 000. This molecular size should allow glomerular filtration of the protein. Four calcium ligands have been demonstrated by electrophoresis-radioautography in dialysed serum (Tejler, unpublished); none of these binders, however, has an electrophoretic mobility indentical to that of the urinary protein. This result does not rule out the possibility that the urinary protein derives from a serum protein existing at a very low concentration in the blood plasma. Whether CaBUP is synthetized in the kidney or transported to it from other sources is not known. Acknowledgements This study was supported, University of Lund.

in part, by a grant from the Medical

Faculty,

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