Isolation and characterization of the neutrophil-binding proteins for platelet-derived adherence-inhibiting factor.
Guinea pig neutrophils adhered to adherence-inhibiting factor (AIF)-coated plastic; the adherence was completely inhibited by the addition of AIF, but partly inhibited by type IV collagen. Binding of 125I-labeled AIF to neutrophils was inhibited by unlabeled AIF, but partly inhibited by type IV collagen. Scatchard analysis showed that neutrophils have two classes of binding sites for AIF, high-affinity binding sites (kd = 5.0 pmol/L) numbering 500 per cell and low-affinity binding sites (kd = 860 pmol/L) numbering 6,400 per cell. Type IV collagen increased the kd of low-affinity binding sites. We have isolated and characterized the AIF-binding sites. We have isolated and characterized the AIF-binding proteins. Using AIF affinity chromatography, the radioactive fraction containing six proteins of molecular mass 45, 63, 87, 90 to 105, 145, and 195 Kd was isolated from 125I surface-labeled neutrophil extracts. This radioactive fraction was further separated into two fractions using type IV collagen affinity chromatography, ie, one fraction was adsorbed on the type IV collagen column and contained the 45-, 63-, and 87-Kd proteins, whereas another fraction was not adsorbed on the column and contained the 45-, 63-, 90- to 105-, 145-, and 195-Kd proteins. To isolate the type IV collagen-binding proteins, 125I surface-labeled neutrophil extracts were applied to a type IV collagen-Sepharose column; the isolated radioactive fraction contained the 45-, 63-, and 87-Kd proteins and bound to an AIF-Sepharose column. Taken together, these results suggest that the AIF-binding proteins, which bind to type IV collagen, are the type IV collagen-binding proteins of neutrophils.
Department of Biochemistry, School of Medicine, Juntendo University, Tokyo, Japan.
K
K
Iwabuchi
I
I
Nagaoka
A
A
Someya
T
T
Yamashita
D000818:Animals; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification; D002448:Cell Adhesion / Q000187:drug effects; D003094:Collagen / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000494:pharmacology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*
D002352:Carrier Proteins; D005353:Fibronectins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003094:Collagen; D008274:Magnesium; D002118:Calcium