Gillian M. Cowell

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250–555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta‐ and intra‐membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

Biosynthesis of intestinal microvillar proteins: Further characterization of the intracellular processing and transport

The effect of tunicamycin on synthesis and intracellular transport of pig small intestinal aminopeptidase N (EC 3.4.11.2), sucrase‐isomaltase (EC 3.2.1.48–10) and maltase‐glucoamylase (EC 3.2.1.20) was studied by labelling of mucosal explants with [35S]methionine. The expression of the microvillar enzymes was greatly reduced by tunicamycin but could be partially restored by leupeptin, suggesting the existence of a mechanism whereby newly synthesized, malprocessed enzymes are recognized and degraded. In the presence of tunicamycin, polypeptides likely to represent non‐glycosylated forms of the enzymes persisted in the Mg2+‐precipitated membrane fraction, indicating that high mannose glycosylation is essential for transport to the microvillar membrane. Treatment of aminopeptidase N and sucrase‐isomaltase with endo F reduced the size of the high mannose forms approximately to those seen in the presence of tunicamycin. The complex forms were also sensitive to endo F but did not coincide with the high mannose forms after treatment, indicating that the size difference cannot alone be ascribed to processing of N‐linked carbohydrate.

Biosynthesis of intestinal microvillar proteins: The intracellular transport of aminopeptidase N and sucrase-isomaltase occurs at different rates pre-Golgi but at the same rate post-Golgi

The kinetics of processing and microvillar expression of aminopeptidase N (EC 3.4.11.2) and sucrose α‐D‐glucohydrolase‐oligo‐1,6‐glucosidase (sucrase‐isomaltase, EC 3.2.1.48 and EC 3.2.1.10) were compared by labelling of pig small intestinal mucosal explants with [35S] lmethionine. The conversion from transient (high mannose glycosylated) to mature (complex glycosylated) form was 1.7‐times slower for sucrase‐isomaltase than for aminopeptidase N, indicating a slower rate of migration from the rough endoplasmic reticulum to the Golgi complex. Likewise, sucrase‐isomaltase appeared in the microvillar fraction at a slower rate than aminopeptidase N. The relative pool sizes of mature and transient forms of both enzymes in intracellular membranes (Mg2+‐precipitated fraction) were determined to obtain information on the relative time, spent pre‐ and post‐Golgi, respectively, prior to microvillar expression. This ratio was 0.24 ± 0.06 (mean ± SD) for sucrase‐isomaltase as compared to 0.40 ± 0.04 (mean ± SD) for aminopeptidase N. Considering the slower rate of pre‐Golgi transport for sucrase‐isomaltase, this indicates that the two microvillar enzymes have rather similar if not identical rates of post‐Golgi transport.

Biosynthesis of intestinal microvillar proteins: Rapid expression of cytoskeletal components in microvilli of pig small intestinal mucosal explants

Using alkaline extraction to separate cytoskeletal and membrane proteins of intestinal microvilli, the kinetics of assembly of these two microvillar protein compartments was studied by pulse‐chase labelling of pig small intestinal mucosal explants, kept in organ culture. Following a 10 min pulse of [35S]methionine, the membrane proteins did not appear in the microvillar fraction until after 40–60 min of chase. In contrast, the cytoskeletal components, of which the 110‐kDa protein and villin were immunologically identified, were expressed in the microvillar fraction immediately after the 10 min pulse. These different kinetics of appearance indicate that the two microvillar protein compartments have separate mechanisms of biosynthesis and microvillar expression.

Microscale purification of proteins by line immunoelectrophoresis: application of the technique in protein biogenesis studies

A small-scale version of line immunoelectrophoresis in combination with immunoprecipitate excision is described as a rapid and convenient technique to purify proteins on a micro scale in biogenesis studies. In the purification and to result in a higher state of purity than an isolation procedure using protein A-Sepharose. Since the method furthermore allows a simultaneous purification of several different protein antigens from the same sample, it may be of interest as an alternative method to other procedures in the purification of proteins on a micro scale.