ISSN:
0730-2312
Keywords:
actin
;
tropomyosin
;
α-actinin
;
reductive methylation
;
microfilament assembly
;
DNase I inhibition
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Actin and tropomyosin, purified from both muscle and brain, and α-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 105 dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield 〉 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0-8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [3H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [14C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.
Additional Material:
8 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240190107
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