Characterization of Geotrichum candidum lipase III with some preference for the inside ester bond of triglyceride

A new form of Geotrichum candidum lipase with a unique positional specificity was found to exist in the culture broth as a minor component together with the well-documented major form. Unlike the major form, which cleaves both the inside and outside ester bonds of triglyceride indiscriminately, the newly isolated form showed some preference for the inside (2-position) ester bond. The new enzyme was also characterized by its own fatty acid specificity, i.e., an outstandingly high activity towards triolein and methyl oleate among the simple triglycerides and fatty acid methyl esters tested. Moreover, the enzyme possesed a specific activity three times as high as the major form. Notable difference in circular dichroism spectra were observed between the two forms, indicating distinct conformational differences. Edman degradation revealed that the N-terminal sequence of the new form differed from that of the major form, thus demonstrating the existence of a novel lipase gene on the chromosome. Correspondence to: A. Sugihara     

Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways.

Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src-family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn-negative and syk-negative B cell lines were established. Syk-deficient B cells abolished the tyrosine phosphorylation of phospholipase C-gamma 2, resulting in the loss of both inositol 1,4,5-trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn-deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.     

Growth regulation of a human mature B cell line, B104, by anti-IgM and anti-IgD antibodies

An EBNA- human B lymphoma cell line, B104, was established. B104 cells express IgD as well as IgM on their surface, which is thought to be a basic characteristic of mature B cells. The growth of B104 cells was inhibited by treatment with a panel of anti-IgM antibodies. Cell cycle analyses revealed that the transition of B104 cells from the G2/M to the G0/G1 phase of the cell cycle was markedly inhibited by treatment with anti-IgM antibodies. Progression of B104 cells to the M phase of the cell cycle was found to be suppressed in the presence of anti-IgM antibodies. In contrast, both the entrance of G0/G1 phase cells into the S phase and the progression of S phase cells to the G2/M phase of the cell cycle did not seem to be inhibited significantly by treatment with anti-IgM antibodies. These results indicate that the mechanism of the inhibition of growth of B104 cells by anti-IgM antibodies is blockage of the transition from the G2 to the M phase of the cell cycle. In contrast to anti-IgM antibodies, anti-IgD antibodies could not cause growth inhibition of B104 cells at all. B cell growth factors such as IL-4 and IL-6 had no effect on the inhibition of growth of B104 cells by anti-IgM antibody. IFN-alpha and -beta, which have no B cell growth factor activity, did increase the number of cells that survived the treatment with anti-IgM antibodies. B104 is an excellent experimental model for the study of the mechanism of signal transduction through sIg as well as the functional difference between sIgM and sIgD.     

Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting.

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.     

Purification and characterization of a Coffea canephora alpha-D-galactosidase isozyme.

Exoglycosidases modify carbohydrate epitopes on glycoproteins and glycolipids. The alpha-D-galactosidase from Coffea canephora is an important exoglycosidase which degrades the human blood group B epitope. Although multiple isozymes have been described, they have never been demonstrably purified and thoroughly characterized. We have developed a technique to purify an isozyme to homogeneity. The isolated enzyme has a molecular weight of 36.7 kDa by SDS PAGE and 34.0 kDa by gel filtration. The isozyme is highly selective for alpha-D-galactosides and inactive against other low molecular weight substrates. It hydrolyzes the the terminal alpha-D-galactosyl residue from the blood group B epitope. Protease activity is below detectable limits. The isozyme has a broad pH optima at 6.3, a pl of 7.03, is unaffected by ionic strength, and is stable at 4 degrees C.     

Ornithine decarboxylase activities of a Shewanella putrefaciens which produces only diamines

Ornithine decarboxylase (ODC) activity in cell extracts of Shewanella putrefaciens was surveyed. The pH dependency of the ODC activity revealed that the bacterium has two different ODC having optimum pH at 8·25 and 6·50. They were considered to be biosynthetic and biodegradative enzymes, respectively. Their activity ratio varied when the bacterium was cultured at pH 7·0 and 6·0. Both ODC activities were inhibited by α-difluoromethylornithine but cell growth was not affected.     

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.     

Missense mutations in exon 5 of the human lipoprotein lipase gene. Inactivation correlates with loss of dimerization.

Most missense mutations of the lipoprotein lipase (LPL) gene identified among LPL-deficient subjects cluster in a segment of the sequence that encodes the catalytic triad as well as functional elements involved in the activation of the lipase at lipid-water interfaces. Consequently, loss of activity may result either from direct alterations of such functional elements or from less specific effects on protein folding and stability. This issue was addressed by examining biochemical properties of four such variants (A176T, G188E, G195E, and S244T) in a heterologous expression system (COS-1 cells). Variant G195E (GGA----GAA) was previously unreported. In all instances, inactive enzyme was recovered in medium, albeit at reduced levels. Cellular synthesis and extracellular degradation were similar to those for wild type, suggesting that reduced secretion resulted from increased intracellular degradation. When cell extracts were subjected to heparin-Superose affinity chromatography followed by elution on a linear salt gradient, all variants exhibited a single, inactive, low affinity immunoreactive peak. By contrast, wild-type enzyme presented an additional, high affinity, active species, which we interpret as homodimeric enzyme. Substitution of the active-site serine (S132A) led to loss of activity but maintenance of the high affinity species. When large amounts of the G188E variant were applied to the column, small but significant amounts of high affinity, active enzyme were recovered. Systematic substitutions at residue 188 showed that only glycine could accommodate structural constraints at this position. We conclude that the mutations examined did not impart lipase deficiency by affecting specific functional elements of the enzyme. Rather, they appear to affect protein folding and stability, and thereby formation and maintenance of subunit assembly.