Inhibition of erythrocyte membrane shape change by band 3 cytoplasmic fragment

The ATP-dependent transformation of crenated white human erythrocyte ghosts into smoothed disc and cup forms is inhibited by the soluble 40-45-kilodalton (kDa) cytoplasmic portion of the major transmembrane protein, band 3. The band 3 fragment was prepared by chymotryptic treatment of inverted vesicles stripped of peripheral proteins. When present at greater than or equal to 0.2 mg per mg membrane protein (ie, greater than or equal to 2 mol fragment per mol endogenous band 3), the fragment significantly reduced the rate of shape change but did not alter the proportion of membranes that were ultimately converted into smoothed forms (greater than 90%). The inhibitory activity of the fragment could not be attributed to contamination of the fragment preparation by actin or proteolytic enzymes. ATP-independent shape transformation was not inhibited. The band 3 fragment may compete with endogenous, intact band 3 for an association with the spectrin-actin network required for ATP-dependent smoothing of crenated membranes.     

Carbonic anhydrase II isoenzyme in rat liver is under hormonal control.

A specific and sensitive radioimmunoassay for rat carbonic anhydrase II (CAII) was developed. Rat livers were perfused with Ringer's solution and the hepatic CAII was measured. Concentrations of CAII in female liver were significantly higher than those in male liver. Castration increased the concentration in male liver, though not to that in females, and diethylstilboestrol treatment of castrated males gave values higher than those in females.     

Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function.

Transfection of a pBR322-based, recombinant plasmid, pAV2, containing the entire adeno-associated virus (AAV) type 2 genome into human 293 cells in the presence of helper adenovirus resulted in rescue and replication of AAV to yield infectious particles. We constructed mutants of pAV2 containing deletions within the AAV sequence. We describe here the phenotypes of these AAV deletion mutants. The results can be summarized as follows. Mutants (cap-) with deletions between map positions 53 and 85 did not synthesize capsid antigen or progeny single-stranded DNA but accumulated normal levels of duplex replicating form DNA. Mutants (rep-) with deletions between map positions 17 and 36 failed to rescue or replicate any AAV DNA. The rep- mutants could be complemented for replicating form DNA synthesis by a cap- mutant. This clearly demonstrates an AAV-coded replication function which is different from the capsid antigen. Other mutants (inf-) with deletions in the region between map positions 40 and 52 synthesized abundant amounts of replicating form DNA and capsid antigen but gave a low yield of infectious particles. This suggests that there may be an additional region of AAV, perhaps within the intron, which is required for efficient particle assembly. This work shows that AAV is genetically complex and expresses at least three clearly different functions.