Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

The human ecto-apyrase gene family consists of five reported members (CD39, CD39-L1, CD39-L2, CD39-L3, and CD39-L4). The family can be subdivided into two groups by conservation of proposed structural domains. The CD39, CD39-L1, and CD39-L3 genes all encode hydrophobic portions in their carboxy and amino termini, serving as transmembrane domains for CD39 and potentially for the other two members. CD39-L2 and CD39-L4 genes encode hydrophobic portions in their amino termini, suggesting that they might encode secreted apyrases. We demonstrate that the CD39-L4 gene encodes the first reported human secreted ecto-apyrase. COS-7 cells transfected with a CD39-L4 expression construct utilizing the naturally occurring leader peptide express recombinant protein outside of the cells. This expression can be blocked by brefeldin A, a chemical that inhibits a step in mammalian secretory pathways. We also demonstrate expression of CD39-L4 message in macrophages, suggesting that the protein is present in the circulation. Furthermore, we show that CD39-L4 is an E-type apyrase, is dependent on calcium and magnesium cations, and has high degree of specificity for NDPs over NTPs as enzymatic substrates. A potential physiological role in hemostasis and platelet aggregation is presented.

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We have identified three novel, rarely expressed human genes that encode new members of the lipid transfer/lipopolysaccharide binding protein (LT/LBP) gene family based on sequence homology. BPI and other members of the LT/LBP family are structurally related proteins capable of binding phospholipids and lipopolysaccharides. Real-time PCR studies indicate that BPIL1 and BPIL3 are highly expressed in hypertrophic tonsils. In situ hybridization analysis of BPIL2 shows prominent expression in skin specimens from psoriasis patients. BPIL1 and BPIL3 map to Chromosome 20q11; thus, these novel genes form a cluster with BPI and two other members of the LT/LBP gene family on the long arm of human Chr 20. BPIL2maps to Chr 22q13. The exon/intron organization of all three genes is highly conserved with that of BPI, suggesting evolution from a common ancestor.

Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.