In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal‐binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide‐containing phytochelatin‐cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP‐binding cassette)‐type transport proteins which includes the mammalian P‐glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1‐expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue‐specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants.

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Phytochelatins (PCs) are metal-chelating peptides produced in plants and some fungi in response to heavy metal exposure. A Cd-sensitive mutant of the fission yeast Schizosaccharomyces pombe, defective in production of a PC-Cd-sulfide complex essential for metal tolerance, was found to harbor mutations in specific genes of the purine biosynthetic pathway. Genetic analysis of the link between metal complex accumulation and purine biosynthesis enzymes revealed that genetic lesions blocking two segments of the pathway, before and after the IMP branchpoint, are required to produce the Cd-sensitive phenotype. The biochemical functions of these two segments of the pathway are similar, and a model based on the alternate use of a sulfur analog substrate is presented. The novel participation of purine biosynthesis enzymes in the conversion of the PC-Cd complex to the PC-Cd-sulfide complex in the fission yeast raises an intriguing possibility that these same enzymes might have a role in sulfur metabolism in the fission yeast S. pombe, and perhaps in other biological systems.

Skin diseases with an inflammatory component, regardless of their etiology, are characterized at some point by the extravasation and subsequent infiltration of leukocytes into the dermal and/or epidermal compartments. This trafficking pattern is determined by a complex series of events whereby the leukocytes interact with cell adhesion molecules (CAM), particularly those induced on endothelial cells following activation with various inflammatory mediators. Vascular CAMs belonging to the selectin family (i.e., P-selectin and E-selectin) are thought to mediate early and reversible events involving leukocyte rolling and margination along the lumenal surface of microvascular cells (post-capillary venules). Certain members of the immunoglobulin supergene family (i.e., VCAM-1 and ICAM-1) regulate later and irreversible steps which lead to firm attachment and subsequent diapedesis of leukocytes. Accumulating evidence suggests that if one blocks the ligand-binding sites between leukocytes and endothelial cells, or inhibits vascular CAM expression, hematopoietic cell extravasation and progressive inflammatory events can be greatly diminished. To identify such inhibitors we developed a cell-based Elisa using the human microvascular cell line HMEC-1. As reported in the present paper, this approach yielded a naturally-occurring, low molecular weight compound which potently inhibits cytokine-induced adhesion molecule expression on cultured endothelial cells, without modulating "house-keeping" proteins.

Microsomes and synaptosomes isolated from calf brain contain a sialidase which cleaves ganglioside substrates. The hydrolysis of [3H]ganglioside GDla by the membrane-bound enzyme has been studied under various conditions.The reaction rate decreased with increasing ionic strength in the incubation mixture, and was progressively enhanced by increasing concentrations of the primary alcohols n-pentanol to n-octanol. This stimulation correlates quantitatively with an increase in membrane 'fluidity' caused by these alcohols as measured by fluorescence depolarization employing 1,6-diphenyl-l,3,5-hexatriene as probe. The dependence of the reaction rate on the amount of enzyme in the incubation mixture was linear only with water-soluble substrates but not with the lipophilic ganglioside substrate. Evidence is presented that lipophilic substrate and enzyme interact mainly within the plane of the membrane presumably by lateral diffusion. Taking this into consideration Michaelis-Menten theory was modified accordingly.As predicted, apparent K,,, values increased linearly with the amount of membrane-bound enzyme added and decreased with the concentration of n-hexanol in the incubation mixture. In the presence of varying n-hexanol concentrations the apparent K,-value decreased with increasing membrane 'fluidity', as measured by fluorescence depolarization of 1,6-diphenyl-l,3,5-hexatriene. On the other hand, as expected, I/ values were not affected by membrane 'fluidity' and increased linearly with the amount of membrane protein.Biological membranes contain enzymes acting on phospholipids and sphingolipids which are constituents of these membranes. During the investigation of two such systems we recognized that the reaction rate was highly dependent on the physical state of the membrane: degradation of sphingomyelin by neutral sphingomyelinase and of ganglioside GDI a by sialidase in neuronal membranes was greatly stimulated by general anesthetics which increase membrane 'fluidity' [ 1,2].Centrifugation studies [1] demonstrated that exogenous lipid substrates are adsorbed to or inserted into the membranes. A model was proposed suggesting an interaction between substrate and enzyme within the membrane by lateral diffusion [l]. Fluorescence depolarization studies with the probe 1,6-diphenyl-I ,3,5-hexatriene indicated a qualitative correlation between increase of membrane 'fluidity and stimulation of lipid degradation in the presence of general anesthetics. In the case of halothane this correlation was demonstrated at clinical concentrations [2,3]. In this paper a quantitative correlation between membrane 'fluidity' and the stimulation of degradation of ganglioside GDla employing the homologous series of primary alcohols will be presented.Attempts to determine kinetic parameters for the system mentioned above met with systematic difficulties, such as nonlinear protein-dependence and strong variations of the Dedicated to Professor Horst Jatzkewitz on the occasion of his 70th birthday.