Pepsin-solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression lgtl 1 libraries. Most of the clones hybridized to either a 3.5-kb or 4.2-kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the a2(VI) or al(V1) chain, respectively. Other clones hybridized to either an 8.5-kb mRNA which very likely encodes the a3(VI) chain or to an unknown 2.0-kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N-terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple-helical domain and the presence of two Arg-Gly-Asp sequences which are potential cell-binding structures.Collagen type VI is a unique member within the family of collagenous proteins [l] and is characterized by a rather short triple helix (length 105 nm, M , = 120000), flanked on each side by a globular domain ( M , z 150000). These monomeric structures have the potential to form well-defined, disulfidelinked dimers and tetramers considered to be the building blocks of microfibrils which are abundant in the extracellular matrix [2]. Collagen VI was originally isolated from pepsin digests of tissues [3], a procedure which results in the loss of more than half of its original mass. Such materials were nevertheless valuable for chemical and electron microscope characterizations [4-71, which indicated that the chain fragments have M , = 40000 -70000. These studies eventually led to the proposal that collagen VI is composed of three constituent chains named al(VI), a2(VI) and a3(VI) [8]. Subsequent studies using antibodies raised against pepsin-solubilized collagen VI demonstrated that the genuine chain constituents are considerably larger, with M , = 110000-140000 19-151. A more recent investigation provided evidence that this size refers only to the al(V1) and a2(VI) chains, as a3(VI) is still larger (Mr = 200000) and is presumably synthesized in precursor form with M , = 250000 [16]. Previously biosynthetic studies demonstrated that a large peptide was closely associated with collagen VI, yet no data had shown that it contained collagenous sequences [17-201. In view of the various attempts to characterize the structure of collagen VI, only little is known about its biological roles [2] which may include the potential for cell-binding [21].A drawback in the studies of intact collagen VI has been the difficulty to isolate the material in amounts sufficient for In an effort to overcome some of these difficulties we have initiated studies on the identificati...

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In the absence of sterol, amphotericin B at 5 x 10-6 M caused maximum marker release from the saturated dipalmitoyl lecithin liposomes, minimum release from the unsaturated dioleoyl lecithin liposomes, and an in-between response from egg lecithin liposomes. Nystatin at 2.5 to 4.0 x 10-M induced appreciable marker release from all three types of sterol-free liposomes. The amphotericin B-and nystatin-induced permeability changes in dipalmitoyl lecithin liposomes were drastically suppressed by the incorporation of cholesterol or stigmasterol (with identical At sterol nuclei), but were unaffected by the incorporation of ergosterol or 5,-7-cholestadien-3,-ol (with identical A5,7 sterol nuclei). The nystatin sensitivity of dioleoyl lecithin liposomes remained low after the incorporation of cholesterol or stigmasterol, but was greatly enhanced by the incorporation of ergosterol or 5,7-cholestadien-3,B-ol. Digitonin, a compound known to interact specifically with membrane sterol, induced marker release from liposomes in proportion to the amount of either cholesterol or ergosterol incorporated; epicholesterol did not sensitize to digitonin. These results lead to the following conclusions: (i) polyene-induced permeability alteration in model membrane systems is effected by the composition of membrane phospholipid fatty acyl chains; (ii) the distribution of double bonds in the sterol nucleus is related to the selective toxicity of the polyenes toward natural sterol-containing membranes; and (iii) polyenes differ in membrane selectivity.The polyene antibiotics are a group of structurally related macrolides which damage certain biological membranes and are used clinically for their fungicidal activity. The widely held concept on the mode of their antifungal action is that all polyene effects are due to their binding to membrane sterols (4,9,12,22). Research efforts during the past decade have been devoted primarily toward the demonstration of direct interaction between polyene and sterol. These studies have been fruitful (10,12,14,15,18). In particular, filipin and cholesterol, the most studied polyene-sterol system, were shown to complex in a stoichiometrically and stereochemically defined manner (14,18).Two questions pertinent to the antimembrane action of the polyenes have not been systematically studied, namely: (i) what is the possible role of other membrane components, such as phospholipid, in conferring polyene-sensitivity; and (ii) what is the basis for the differential toxicity of polyenes versus membranes containing various sterols? In the present work, the effect on several membrane systems of two clinically useful polyenes, amphotericin B and nystatin, were studied as part of an effort to answer the questions. In this paper, our results with lecithin liposomes are presented; a brief preliminary report of some of these data has appeared (5). In the accompanying paper (6) our studies of selected natural membranes are presented.Previous studies on polyene-model membrane interaction (10,14,15,18)

The effect of polyene antibiotics on Candida albicans , human erythrocytes, and Acholeplasma laidlawii was studied. The results sustain the observations made with lecithin-sterol liposomes. The distribution of double bonds in the membrane sterol nucleus appears to be of major importance in conferring polyene susceptibility; those sterols with the ergosterol nucleus are far more effective than those with a nucleus similar to cholesterol. Different polyenes vary in their membrane selectivity. The clinical implications of these observations are discussed.