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An abnormal asubunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the asubunit (aI T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the aI domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of aI T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin asubunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of a I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of a I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kin-dreds studied demonstrated polymorphism in the spectrin a II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant a III domain in spectrin that has not previously been described. We conclude that the abnormality in the a I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the a subunit that is affected.

Hereditary pyropoikilocytosis (HPP) and hereditary elliptocy-tosis are closely related, congenital disorders of the red blood cell usually associated with defective spectrin selfassociation and abnormal limited tryptic digestion of the N-terminal al domain of spectrin. Enhanced cleavage by trypsin of spectrin from affected individuals at arginyl residue 45*and lysyl residue 48**The numbering system is based on the translated amino acid sequence of a-spectrin cDNA,33which includes six additional amino acid residues compared with previous reports that numbered residues from the first amino acid of the αI/80-Kd domain.4frequently yields increased amounts of an αl/74-Kd fragment at the expense of the normal α l/80-Kd parent fragment. Limited tryptic digestion of three unrelated individuals with HPP showed the a 1/74 defect. To ascertain the molecular defect responsible for the abnormality, the structure of exon 2 of the o-spectrin gene was examined. Genomic DNA from the subjects was amplified by the polyermase chain reaction using primers flanking exon 2. Restriction endonuclease digestion of amplified products showed the loss of the Wndlll site at codons 47 and 48 in one allele of subject 1 and abolished the Ahall site at codons 27 and 28 in one allele of subjects 2 and 3. Nucleotide sequence analysis of subcloned amplified DNA from the HPP subjects showed three novel amino acid substitutions. In subject 1 (a black individual), a single base substitution (AAG → AGG) at codon position 48 changes amino acid residue lysine to arginine. In subject 2 (a white individual), a single base substitution (CGT → AGT) at codon 28 changes arginine to serine. In subject 3 (a black individual), a different base substitution at position 28 (CGT → CTT) changes arginine to leucine. These mutations occur at positions of the al domain where other mutations have also been described, indicating that the normal residues at these positions play an important role in spectrin dimer self-association and thus, in membrane stability.

The metabolism of the ribosomal and soluble protein components of Aerobacter aerogeneswas examined during its incubation in a Mg++-deficient medium. Bacteria were exposed to leucine-H3during the exponential growth period preceding Mg++starvation, and extracts were prepared after intervals of starvation and were centrifuged through gradients of sucrose to separate ribosomal from soluble proteins. Ribosomal proteins synthesized during the preceding exponential growth were slowly lost from the ribosomes; after 8 hr of starvation, few, if any, sedimented with ribosomes. Losses of total protein, together with the known rate of ribosome decay during Mg++starvation, suggested that these ribosomal proteins are ultimately degraded to acid-soluble products and account for all protein lost by the starving cells. These conclusions were supported by studies of Mg++starvation in a uracil-requiring strain of A. aerogenes: during uracil starvation a smaller fraction of the proteins synthesized were ribosomal, and the fraction of protein which subsequently decayed during Mg++starvation was correspondingly less. During recovery from Mg++starvation, proteins, lost from disintegrated ribosomes, were not detectably reutilized into new particles even before their degradation to acid-soluble products was complete. Synthesis of soluble proteins continued for more than 24 hr of starvation at a rate per milliliter close to 45% of the instantaneous rate per milliliter of the exponentially growing bacteria at the time Mg++was removed. This value agreed with that found previously for synthetic rates of deoxyribonucleic acid, transfer ribonucleic acid, and ribosomal ribonucleic acid during starvation relative to rates during exponential growth.