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A protein-NeuAc complex involved in colominic acid biosynthesis has been identified in membrane preparations of Escherichia coli K-235. This compound had an Mr (estimated by SDS/polyacrylamide-gel electrophoresis and autoradiography) of about 100,000 and played the role of an 'initiator' or 'primer' (endogenous acceptor) in the synthesis of the whole polymer. Incubations of E. coli membranes with CMP-[14C]NeuAc (CMP-N-[14C]acetylneuraminic acid) pointed to the existence of a protein fraction (primer acceptor) that linked residues of sialic acid (N-acetylneuraminic acid, NeuAc) up to a maximal size, later releasing them as low-Mr sialyl polymers (LMrS, Mr less than 10,000). In the presence of colominic acid (final acceptor) the radioactivity linked to the protein quickly decreased, appearing stoichiometrically bound to the whole polysaccharide. When membrane preparations were previously digested with Streptomyces proteinase or de-activated by heating (80 degrees C, 10 min), no incorporation of labelled NeuAc into trichloroacetic acid-insoluble material was detected. These results suggested that colominic acid molecules are synthesized while they are bound to a proteinaceous acceptor that is subsequently excised in the presence of colominic acid, generating the native protein. The antibiotic tunicamycin inhibited the biosynthesis of colominic acid, affecting the synthesis of this protein-(NeuAc)n intermediate. All these results are described here for the first time.

The membrane-bound sialyltransferase obtained from Escherichia coli K-235 grown in a chemically defined medium (ideal for colominic acid production) was studied. The in vivo half-life calculated for this enzyme was 20 h. Kinetic tests revealed (at 33 "C and pH 8.3) hyperbolic behaviour with respect to CMP-NeuSAc (K, 250 pM) and a transition temperature at 31.3"C. The enzyme was inhibited by NH:, some divalent cations and by several agents that react with thiol groups. Detergents and fatty acids also inhibited the sialyltransferase activity. In vitro synthesis of colominic acid is strongly inhibited by CMP by blocking the incorporation of [ 14C]Neu5Ac into a protein-complex intermediate and therefore into free polymer. CDP and CTP also inhibited (91 YO and 84%) this enzyme activity whereas cytosine and cytidine had no effect. In Escherichia coli K-235 the synthesis and assembly of colominic acid (an a2+8 hoinopolymer of NeuSAc) has been shown to be mediated by a membrane-associated sialyltransferase complex which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products or into a lipid fraction [S, 9, 111. Although the biosynthesis of colominic acid has been extensively studied [6-121, little is known about the regulation and metabolic effectors involved Correspondence fo J. M. Luengo,

This article is available online at http://www.jlr.org Supplementary key words hormone-sensitive lipase • cholesterol esters • steroidogenesis • spermatogenesis • fertility • sterility Spermatogenesis occurs in a series of proliferative and differentiation stages, which can be subdivided into mitotic, meiotic, and spermatogenic phases. Each phase is characterized by distinct cell types (spermatogonia, spermatocytes, and spermatids, respectively) ( 1, 2 ). Pathologic, experimental, and natural seasonal arrest of spermatogenesis are all associated with increases in lipid droplets ( 3, 4 ), indicating a close relationship between fertility and changes in lipid metabolism during spermatogenesis ( 5 ). A constant supply of cholesterol is required within Leydig cells to serve as precursor for the synthesis of steroid hormones (steroidogenesis) ( 6, 7 ), whereas in the seminiferous tubules, cholesterol is involved in germinal cell differentiation to spermatozoids (spermatogenesis or gametogenesis) ( 8 ).There is also considerable evidence indicating that cholesterol is required for the development of gametes and fertility in both sexes. Disruption of the DHCR24 gene, which encodes the cholesterol biosynthetic enzyme desmosterol reductase, causes infertility in male mice ( 9 ). In addition to cholesterol, sterol Abstract There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterifi ed cholesterol by hormone-sensitive lipase (HSL). HSL is critical