Apolipoprotein A-l-contalnlng llpoprotelns (high density llpoproteins, HDL) can be separated Into two subtractions, which have pre-beta and alpha electrophoretic mobilities, respectively. These fractions differ In both composition and structure. Some preparations of pre-beta-mlgratlng HDL, but not alpha-migrating HDL, were found to contain two polypeptldes with M r of approximately 26 and 14 kDa, which are scission products of apolipoprotein (apo) A-l. They are recognized by monospeclflc antibodies to apo A-l and have ^-terminal sequences Identical to those of mature apo A-l. This proteolytic scission of apo A-l occurs primarily after venlpuncture. Immediate addition of protease Inhibitors minimized the appearance of the fragments In plasma. To study the relative susceptibilities of pre-beta and alpha HDL to proteolysis, the llpoprotelns were Incubated In vitro with plasmln. The apo A-l In pre-beta HDL was extensively degraded, but that In alpha-migrating HDL was degraded to a much lesser extent, Indicating that the appearance of apo A-l fragments In pre-beta HDL was due to enhanced sensitivity to proteolysis. To varying degrees, thrombln, kallikreln, elastase, arglnlne C endoprotease, and chymotrypsin also appear to cleave pre-beta HDL faster than alpha HDL Most of the proteases generated a 12 to 14 kDa peptlde fragment under conditions of limited cleavage. These results suggest that the conformatlonal state of apo A-l In pre-beta-mlgratlng HDL or Its spatial relationship to llplds Is significantly different from that of apo A-l In alpha-migrating HDL. Furthermore, this conformation of apo A-l appears to expose a protease-sensltive region near the midpoint of the sequence. Finally, when studies of pre-beta HDL are undertaken, care should be taken to prevent proteolytic degradation of this particle. (Arteriosclerosis 10:25-30, January/February 1990) H igh density lipoproteins (HDL) are a collection of subspecies with different compositions and properties. One such HDL subpopulation has pre-beta electrophoretic mobility (pre-beta HDL).1 This group of lipoproteins has a composition that is distinctly different from the bulk of alpha-migrating HDL.2 Pre-beta HDL also appear to possess structural properties that distinguish them from the bulk of HDL.2 The in vitro proteolytic degradation of apolipoprotein (apo) A-l has been demonstrated for the enzymes plasmin, 34 thrombin, 4 trypsin, 5 ' 6 and chymotrypsin. 5 The degradation of apo A-l on intact HDL has also been studied, 8 -6 allowing the identification of exposed and protected regions. But no apparent differences in the proteolytic degradation of HDL fractions HDLj, and HDU have been determined. We describe here the enhanced sensitivity of the apo A-l in pre-beta HDL to in vitro proteolytic cleavage when compared to alpha HDL Methods Isolation of PlasmaPlasma was isolated from freshly drawn venous blood by centrifugation at 10 000 g for 30 minutes at 4°C in the presence of preservatives and inhibitors, 0.04% ethylenediaminetetraacetjc acid (EDTA), 0.05% NaN 3 , ...

In anthers, the tapetum synthesizes and stores proteins and flavonoids, which will be transferred to the surface of adjacent microspores. The mechanism of synthesis, storage, and transfer of these pollen-coat materials in maize (Zea mays) differs completely from that reported in Arabidopsis (Arabidopsis thaliana), which stores major pollen-coat materials in tapetosomes and elaioplasts. On maize pollen, three proteins, glucanase, xylanase, and a novel protease, Zea mays pollen coat protease (ZmPCP), are predominant. During anther development, glucanase and xylanase transcripts appeared at a mid developmental stage, whereas protease transcript emerged at a late developmental stage. Protease and xylanase transcripts were present only in the anther tapetum of the plant, whereas glucanase transcript was distributed ubiquitously. ZmPCP belongs to the cysteine protease family but has no closely related paralogs. Its nascent polypeptide has a putative amino-terminal endoplasmic reticulum (ER)-targeting peptide and a propeptide. All three proteins were synthesized in the tapetum and were present on mature pollen after tapetum death. Electron microscopy of tapetum cells of mid to late developmental stages revealed small vacuoles distributed throughout the cytoplasm and numerous secretory vesicles concentrated near the locular side. Immunofluorescence microscopy and subcellular fractionation localized glucanase in ER-derived vesicles in the cytoplasm and the wall facing the locule, xylanase in the cytosol, protease in vacuoles, and flavonoids in subdomains of ER rather than in vacuoles. The nonoverlapping subcellular locations of the three proteins and flavonoids indicate distinct modes of their storage in tapetum cells and transfer to the pollen surface, which in turn reflect their respective functions in tapetum cells or the pollen surface.