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Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons
We have characterized the interaction of bovine pancreatic deoxyribonuclease I (Dnase I) with the filamentous (F-) actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H] DNA was used to assay Dnase I. We found that Dnase I bound to a homogenous class of =2.4 X 10 4 sites/skeleton with an association rate constant of approximately 1 X 10 6 M -1 S -1 and a Kc of 1.9 X 10 -9 M at 20 C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The Dnase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to Dnase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested.
Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerizatin of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely ingibited by 35 nM DNase I but not be 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament. |
Research Data
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| Ending
Year:
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1988 |
| Design:
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| Study
Type:
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Funded-Government |
| Theoretical
Framework:
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none |
| Description
of Sample:
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| Sample
Size:
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| Number
of Groups:
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| Sampling
Plan:
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Random selection |
| Gender:
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Mixed |
| Minimum
Age:
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| Maximum
Age:
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| Data
Collection Settings(s):
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Laboratories |
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Primary Investigator
Janice L. Podolski, PhD
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P.I. Institution Name
Rush University College of Nursing
Title
Assistant Professor
Contact Address
600 S. Paulina, Suite 1080
Chicago, IL, 60612
USA
Contact E-mail
jpodolski@rushu.rush.edu
Contact Telephone
312.942.6103
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Secondary Investigators
T.L. Steck
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