No abstract

Purified liver lysosomes, prepared from rats previously injected with Triton WR-1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity.The intralysosomal sialidase was solubilized from the mitochondrial-lysosomal fraction of rat liver and partially purified by Sephadex G-200, or Sephadex G-200 followed by CM-cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60000 k 5000 by gel filtration; it hydrolyzed a variety of sialooligosaccharides, those containing (a2+ 3)sialyl linkages being better substrates than those with (a2+6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and transferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin.In contrast to the intralysosomal silaidase, the sialidase partially purified from rat liver cytosol by (NH,),SO, fractionation followed by chromatography on DEAE-cellulose and CM-cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maximally active at pH 5.8 and had a relative molecular mass of approximately 60000. It also hydrolyzed gangliosides but not submaxillary mucin.

A full-length cDNA encoding rat type 2C (IA) protein phosphatase was isolated from a kidney cDNA library. The cDNA was identified by screening the library with oligonucleotides based on a partial amino acid sequence determined from purified rat liver phosphatase. This clone is 2.35 kilobase pairs long and has a single extended translation reading frame that predicts a 382-amino acid protein of 42,416 daltons. The deduced amino acid sequence contains segments corresponding to three peptides from rat liver type 2C protein phosphatase and two peptides from rabbit skeletal muscle type 2C phosphatase. Rat kidney type 2C protein phosphatase is distantly related to yeast adenylate cyclase but is not related to the catalytic subunits of two other protein phosphatases (types 1 and 2A).Protein phosphorylation plays a key role in the regulation of cellular functions. Although most studies focus on protein kinases, the importance of protein phosphatases in the phosphorylation/dephosphorylation cycle has been well documented (1). In this context, we previously reported the purification and characterization of three types of cytosolic rat liver phosphatases, types IA, IB, and II (2-5). These enzymes were later independently identified and named types 2C, 2A1, and 2A2, respectively (6). Since this later nomenclature is more widely used, we have adopted it in this report. The type 2C enzyme (Mr 42,000-48,000) is monomeric and requires Mg2+ for activity (4, 5). Types 2A1 and 2A2 are heterooligomeric and have two common subunits termed a (Mr 35,000) and ,8 (Mr 69,000); the a subunit is the catalytic moiety (2, 3). Type 2A1 has a third subunit, y (Mr 58,000). The functions of the P and y subunits are unknown. Although the type 2C enzyme was originally thought to be specific for glycogen synthase (7,8), it was later shown that this enzyme is active with a wide variety of substrates, at least in vitro (5,6). Neither the role of this enzyme in dephosphorylation in vivo nor the mechanism of its regulation has been described to date. To further understand the biology of the type 2C phosphatase, we undertook the molecular cloning of its mRNA.t# MATERIALS AND METHODS Isolation and Sequence Analysis of Peptides. Type 2C protein phosphatase was purified from rat liver as described (4, 5). The purified protein (100 ttg) was digested with trypsin and the resulting peptides were purified by HPLC (9). The peptides were sequenced by automated Edman degradation (10).Oligodeoxynuclegtide Synthesis. Oligonucleotides encoding regions of amino acid sequence were designed using the consensus rules ofLathe (11) and synthesized on a BioSearch 8600 synthesizer.Cloning. The rat liver and kidney libraries were constructed and screened as described (12, 13). The EcoRI inserts were subcloned into pGEM-series plasmids (Promega Biotec) and DNA sequence was determined by the dideoxy chain-termination technique of Sanger et al. (14) after construction of a series of nested deletions. In some cases, the dGTP analogue 7-deaza-dGTP (Toyoba, Osaka, Japan...

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by CAMPdependent protein kinase, protein kinase C and Ca2 +/calmodulin-dependent protein kinase 11. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type I1 keratin 8 (molecular mass 55 kDa; basic type) in a 1 : 1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by CAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2 c calmoddin-dependent protein kinase I1 were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.

Ganglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2,150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited M = 70,000 by gel filtration. Sialidase I hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase I reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.