Molecular and functional identification and purification of complement component factor D from urine of patients with chronic renal failure.
Urine proteins of normal subject and patients with impaired renal function were analyzed by two-dimensional polyacrylamide gel electrophoresis. As a result, a clear spot was detected specifically in urine from patients with obvious renal dysfunction. The isoelectrical point of this unique spot was pH 7.1-7.2 and the flow-rate (Rf) was 0.50-0.55 as that of albumin was 1.0. Partial amino acid sequence analysis revealed that the NH2-terminal to 22nd amino acid sequence was identical with that of complement factor D. We purified 22 mg of this protein (factor D) from 5000 ml of urine from a patient on hemodialysis by three chromatographic steps using DEAE-Sephadex A-50 and Sephacryl S-200. The purified urine factor D gave a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis at the position of 23 kD, and displayed normal factor D hemolytic activity. The concentrations of factor D estimated by hemolytic assay were 1.9 micrograms/ml of normal serum, less than 0.1 microgram/ml of normal urine, 15 micrograms/ml of patient serum and 50 micrograms/ml of patient urine.
Department of Internal Medicine, Nagoya University School of Medicine, Branch Hospital, Japan.
T
T
Miyata
O
O
Oda
R
R
Inagi
S
S
Sugiyama
A
A
Miyama
K
K
Maeda
I
I
Nakashima
N
N
Yamanaka
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001681:Biological Assay; D002850:Chromatography, Gel; D003166:Complement Activating Enzymes / Q000652:urine*; D011416:Complement Factor D / Q000652:urine*; D015180:Electrophoresis, Gel, Two-Dimensional; D006461:Hemolysis; D006801:Humans; D007676:Kidney Failure, Chronic / Q000652:urine*; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000652:urine*
D000596:Amino Acids; D003166:Complement Activating Enzymes; D012697:Serine Endopeptidases; C493488:CFD protein, human; D011416:Complement Factor D