The salient pathological feature of Azhelmer (1-4).Radloiodination. Peptides containing tyrosine were radiolabeled by oxidative radioiodination using Na125I and chloramine T and separated from free iodide by reverse-phase adsorption. Peptides not containing tyrosine were first acylated with the N-hydroxysuccinimide ester of 4-hydroxyphenylpropionic acid and then oxidatively radioiodinated as above. Labeled peptides containing methionine were then reduced from sulfoxide to native form with 2-mercaptoethAbbreviations: AD, Alzheimer disease; ,BA4, ,B-amyloid peptide; RP-HPLC, reversed-phase HPLC; 125I-PA4-(1-40), 1251-labeled ,BA4-(1-40). tTo whom reprint requests should be addressed. 5462The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

ZFY, a sex-related Zn-finger protein encoded by the human Y chromosome, is distinguished from the general class of Zn-finger proteins by the presence of a two-finger repeat. Whereas odd-numbered domains and linkers fit a general consensus, even-numbered domains and linkers exhibit systematic differences. Because this alternation may have fundamental implications for the mechanism of protein-DNA recognition, we have undertaken biochemical and structural studies of fragments of ZFY. We describe here the solution structure of a representative nonconsensus (even-numbered) Zn finger based on 2D NMR studies of a 30-residue peptide. Structural modeling by distance geometry and simulated annealing (DG/SA) demonstrates that this peptide folds as a miniglobular domain containing a C-terminal beta--hairpin and N-terminal alpha-helix (beta beta alpha motif). These features are similar to (but not identical with) those previously described in consensus-type Zn fingers (derived from ADR1 and Xfin); the similarities suggest that even and odd ZFY domains bind DNA by a common mechanism. A model of the protein-DNA complex (designated the "jumping-linker" model) is presented and discussed in terms of the ZFY two-finger repeat. In this model every other linker is proposed to cross the minor groove by means of a putative finger/linker submotif HX4HX3-hydrophobic residue-X3. Analogous use of a hydrophobic residue in a linker that spans the minor groove has recently been described in crystallographic and 3D NMR studies of homeodomain-DNA complexes. The proposed model of ZFY is supported in part by the hydroxyl radical footprint of the TFIIIA-DNA complex [Churchill, M.E.A., Tullius, T.D., & Klug, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5528-5532].

Octapeptides ntheized from D amino adds were absorbed from the i id excreted In urine of al rats drinking 5% glucose/1% nineconingthe 12'I-ladbed peptides at 0.1-25 ng/dI. The rats fluid at the rate of about 20 ml/hr and produced urine at 15 ml/hr for several hours during the nocturnal feeding period. Sixty- RATIONALE OF EXPERIMENTS Rats provided with diluted milk or with 5% glucose as the sole source of calories will drink >50% of their body weight during each nocturnal feeding period, producing correspondingly large volumes of urine (11,12). Inert solutes added to the ingestate may be recovered from the urine, thereby providing a minimum estimate of their absorption from the gastrointestinal tract. For example, creatinine or mannitol ingested with 5% glucose can be recovered from urine or other body fluids with a yield of 50-65% in experimental animals (12) and >80%6 in man (13). Experiments of this type provide strong evidence that solutes the size of hexoses or amino acids can be absorbed paracellularly under conditions where fluid absorption and junctional permeability of the small intestine have been increased. In the present investigation we applied this technique to peptides synthesized from D amino acids, anticipating that such molecules would not be recognized by peptidases or transport proteins. For this purpose we synthesized three such peptides having approximately the same molecular weights (octapeptides) but with differing lipid solubilities and net electrical charge (Table 1). Tyrosine (Y) was included in the sequence to permit labeling with 125I, but otherwise the composition and sequences were arbitrary and bear no known relation to biologically active peptides.In preliminary experiments it was found that 30-60% ofthe 125I label from all three of the peptides listed in Table 1 was recovered in urine after ingestion with glucose. However, we soon discovered that the two most lipophilic peptides (DP1 and DP2) were partially converted to polar compounds by the liver and were subsequently recycled in the enterohepatic circulation. Passage of these polar compounds through sizing columns or Bio-Gel Pa (BioRad)] indicated that alteration of the original peptides in liver did not involve major changes of molecular weight. Only the lipid-insoluble peptide DP3 was excreted in urine unaltered, thus providing an uncomplicated test of the hypothesis presented in the Introduction. For this reason the present communication will be concerned mainly with DP3, leaving a detailed description of the more complex behavior of DP1 and DP2 for a subsequent report. METHODSAnimals and Experimental Protocols. Adult male or female rats were maintained on chow pellets at -15 g/day with water ad libitum. This food intake was sufficient to maintain body weight, but the limitation offood made the rats eager to ingest 5% (wt/vol) glucose when they were placed in stainless steel metabolism cages for administration of fluids and collection of feces. Fluid was supplied from weighed bottles, and in Abbreviation: TFA, trif...

The effects of ergosterol, yeast's natural sterol, on cell cycling and a protein kinase antigenically related to pp6O' were examined in a sterol auxotroph ofSaccharomyces cerevisiae. Sterol-depleted cells accumulate in an unbudded, G1 state. Cell budding and proliferation are reinitiated upon addition of nonlimiting ergosterol or cholesterol with trace ergosterol, whereas cholesterol or trace ergosterol alone is less effective. Stimulation of a protein kinase associated with immune complexes of yeast protein and anti-pp60v1 shows a positive correlation with exit from the G1 phase following ergosterol addition. Ergosterol-stimulated cells also demonstrate an increase in phosphatidylinositol kinase activity. The data suggest that hormonal levels of ergosterol (effective concentration, 1 nM) participate in a signaling process associated with a protein kinase possibly involved in yeast cell cycle control.In the budding yeast Saccharomyces cerevisiae nutritional signals normally control cell growth and division. When yeast cells stop growing in response to nutritional deprivation they arrest in the unbudded, G1 phase of the cell cycle (1). The isolation of many temperature-sensitive cell division cycle (cdc) mutants has led to a better understanding of the regulation of yeast cell proliferation and has resulted in the description of a G1-phase "start" event that is controlled by a number of genes (2). One of these genes, CDC28, has been cloned and sequenced (3). The CDC28 gene product shares homology with a number of mammalian protein kinases, among these a vertebrate cAMP-dependent protein kinase, as well as viral oncogenes such as v-mos and v-src (3). The CDC28-encoded protein has now been shown to possess a protein kinase activity, as has a related cdc2+-encoded protein in the fission yeast (4, 5). Moreover, a decrease in the activity ofthe cdc2-encoded protein correlates with cell cycle arrest upon nitrogen starvation in Schizosaccharomyces pombe (5). cAMP-dependent protein kinase and the RAS gene product have also been implicated in growth control in yeast (6). Interestingly, an association has been demonstrated between the activities of the pp60vsrc tyrosine kinase and phosphatidylinositol (PtdIns) kinase in Rous sarcoma virustransformed chicken embryo fibroblasts (7). Although the role of the polyphosphatidylinositol phosphates in yeast physiology has yet to be determined (8-10), we have observed a marked ergosterol-mediated stimulation of the turnover of these lipids that correlates with an enhanced rate of cell proliferation (11).Studies Gl-phase cell cycle arrest (sterol starvation) or G1 release and cell proliferation (sterol addition). This analysis has led to the identification of a protein kinase having antigenic homology with the transforming protein of the Rous sarcoma virus and whose activity is positively regulated by a trace amount of ergosterol. To our knowledge, evidence for the existence of a protein kinase possibly involved in yeast cell cycle control, whose activity is exquisitely sens...

Mycoplasma capricolum, a procaryotic sterol auxotroph, shows optimum growth on cholesterol and substantial growth on lanosterol. The effect of the two sterols on membrane fluidity, their ability to support growth on a broad range of fatty acid combinations, and a possible synergism of low amounts of cholesterol and high amounts of lanosterol were studied. When the cholesterol content of M . capricolum membranes rose from 14 to 28 mol % of the total lipid, their microviscosities increased from 51 = 3.3 to 4.5, whereas membranes containing 14-23 mol % lanosterol showed a constant microviscosity value of = 3.1. Arrhenius plots of the microviscosity values of membranes rich in cholesterol R e c e n t studies in this laboratory have disclosed marked differences in the membrane behavior of cholesterol and lanosterol, a trimethyl-substituted precursor of cholesterol. Incorporated into model membranes such as phosphatidylcholine vesicles, lanosterol, in contrast to cholesterol, neither raises the microviscosity nor reduces the exit of vesicle-entrapped glucose . Carbon-13 nuclear magnetic resonance studies have shed light on the physical basis for these differences; lanosterol is much less immobilized in lecithin lipid bilayers than cholesterol (Yeagle et al., 1977).were linear over a 40 "C range, but those rich in lanosterol exhibited prominent discontinuities at 20 and 25 "C. Cholesterol allowed the cells to grow on media containing a wide variety of fatty acid supplements, whereas lanosterol supported growth only with certain fatty acid combinations. Finally, low levels of cholesterol unable to support the growth of M . capricolum produced a synergistic effect on growth when combined with lanosterol. The results demonstrate the superiority of cholesterol compared to lanosterol as a membrane sterol and suggest a possible role for cholesterol in addition to or other than to regulate bulk membrane fluidity.In eucaryotic sterol auxotrophs lanosterol has also been shown to be incompetent as a substitute for cholesterol (Clark & Bloch, 1959; Chang et al., 1977). Unexpectedly, however, Mycoplasma capricolum, a sterol-requiring procaryote, was found to grow moderately well on lanosterol, without modifying it, as well as on cholesterol (Odriozola et al., 1978). This relatively broad sterol specificity offers the opportunity for evaluating the possibility of multiple sterol functions in membranes and the importance of membrane physical state as a determinant of mycoplasma growth. To this end, we have (a) compared the relative ability of cholesterol and lanosterol to modulate membrane fluidity and (b) examined the effect of supplementing lanosterol-containing growth media with low levels of cholesterol. We find that quantities of cholesterol, inadequate by themselves, will, when combined with lanosterol, support mycoplasma growth in a synergistic manner. We also