We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

The metabolism and distribution of newly synthesized aggrecan present in the extracellular matrix of intact explant cultures of mature articular cartilage was investigated with respect to type VI collagen-stained chondrons. Using biochemical, autoradiographical and novel confocal immunohistochemical techniques it was shown that aggrecan exists as a number of distinct pools that are located within the extracellular matrix of the tissue. The first was identified as a pool of high specific radioactivity, much of which appeared in the medium one day after incubation with radiolabeled sulfate. Of the radiolabeled aggrecan remaining within the extracellular matrix, three pools were differentiated on the basis of time and location within the extracellular matrix. One pool was resident within the pericellular microenvironment associated with the chondron, one migrated into the territorial matrix adjacent to the chondron and one was sequestered long term in the interterritorial matrix. Analysis of the kinetics of loss of radiolabeled aggrecan macromolecules present in the region of matrix defined by the chondron suggests that this pool rapidly turns over and is a precursor to the pools of aggrecan present in the territorial and interterritorial matrix. There were marked differences in the distribution of newly synthesized aggrecan present in these regions of the extracellular matrix in explant cultures maintained with or without fetal calf serum. In the absence of serum, more of the newly synthesized aggrecan moved into the territorial and interterritorial matrix indicating that the presence of serum in the culture medium influenced the tissue distribution of aggrecan. This work indicates that the pericellular microenvironment of the chondron plays an important role in the retention and maturation of aggrecan prior to the sequestration of aggrecan complexes into the functional load bearing matrices of adult articular cartilage.

1. Whole scrapings of rat intestinal mucosa were incubated with carrier-free sodium [(35)S]sulphate. Radioactivity was found in S-sulphocysteine and to a small extent in S-sulphoglutathione. 2. Whole scrapings of rat intestinal mucosa incubated with carrier-free sodium [(35)S]sulphate and oxidized glutathione formed S[(35)S]-sulphoglutathione as the main radioactive product. The amount of S[(35)S]-sulphocysteine formed was considerably lower than in a control that contained no oxidized glutathione. 3. The supernatant fraction of homogenates of rat intestinal mucosa catalyses the NADPH-dependent reduction of adenosine 3'-phosphate 5'-sulphatophosphate to inorganic sulphite. NADH or GSH fail to replace NADPH as reducing agents. 4. The formation of inorganic [(35)S]sulphite from inorganic [(35)S]-sulphate may account for the incorporation of [(35)S]sulphate into S-sulphoglutathione by the small intestine of the rat in vivo and in vitro.

A method of peptide "fingerprinting" has been developed allowing the separation of the majority of the tryptic peptides of purified proteoglycan subunit from bovine nasal cartilage. When this preparation was reacted with 0.2 M sodium sulfite at pH 11.5, beta-elimination of the substituted glycosaminoglycans and O-linked oligosaccharides and the quantitative addition of sulfite occurred in the serine and threonine residues of the linkage region. After elimination-addition studies with sodium [35S] sulfite, 6 radiolabelled linkage peptides were isolated by 2-dimensional "fingerprinting." Five of these peptides were derived from a section of the protein core in which each [35S] cysteic acid residue was separated by an average of 6-10 amino acid residues. Apart from [35S] cysteic acid, the predominant amino acids in the attached peptides were glycine and glutamic acid (or glutamine), suggesting that a combination of these amino acids in the nascent protein core may be important for the initiation of glycosaminoglycan chains during proteoglycan biosynthesis.