Regulation of lysosomal α-mannosidase-1 synthesis during development in Dictyostelium discoideum

The cellular specific activity of lysosomal α-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. α-Mannosidase-1 is composed of two subunits (M_r = 58,000 and 60,000) which are derived from a common precursor polypeptide (M_r = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t_1/2 < 10 min). This indicates that the developmental accumulation of α-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional α-mannosidase-1 mRNA during development.     

α-Mannosidase-2: A developmentally regulated enzyme in Dictyostelium discoideum

A previously undected isozyme of α-mannosidase was observed in several independent mutant strains of Dictyostelium discoideum selected for the absence of the major isozyme, α-mannosidase-1. The activity in the mutant strains, α-mannosidase-2, differs from the major isozyme with respect to pH optimum, substrate affinity, sensitivity to inhibition by l-cysteine, and is particulate bound. The enzyme can be solubilized by treatment of the extract with nonionic detergents. α-Mannosidase-2 begins to accumulate only after 12 hr of development and reaches a peak specific activity of about a tenth of that of α-mannosidase-1 during culmination. The increase in specific activity of α-mannosidase-2 is blocked by either cycloheximide or actinomycin D, drugs known to inhibit protein and RNA synthesis, respectively, and probably results from accumulation of de novo synthesized enzyme. α-Mannosidase-2, therefore, provides a convenient marker enzyme for biochemical differentiation during the pseudo-plasmodial stage.     

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25^oC, the major and minor components of the E3 isozyme catalyzed the reaction with V_max of 1.1 and 0.8 μmol NADH min⁻¹ mg⁻¹ protein, respectively, compared to 0.067 and 0.060 μmol NADH min⁻¹ mg⁻¹ protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K_m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K_i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.     

Esterase isozyme polymorphism, specific and nonspecific esterase, syngenic lines development and natural occurrence of a thermostable esterase in the tropical silkworm Bombyx mori L.

Esterase isozyme polymorphism was documented for digestive juice and haemolymph of the tropical multivoltine silkworm, Bombyx mori L., breed CB5 (GP) and its syngenic lines (CB5Lm^e-1, CB5Lm-2 and CB5Lm-5) using α- and β-naphthylacetate separately as nonspecific substrates (Ogita, Z., Kasai, T., 1965. Genetico-biochemical analysis of specific esterases in Musca domestica. Jpn. J. Genet. 40, 173–184). Polymorphism existed in the isozyme pattern of α-esterase with two or three bands in digestive juice and three to five bands in haemolymph. No polymorphism was observed in β-esterase isozyme pattern having four bands in digestive juice and two bands in haemolymph. During the course of esterase isozyme studies, the presence of some specific α-esterase bands (Est-1, 4 and 5) in haemolymph and β-esterase bands (Est-1, 2 and 3) in digestive juice were observed. But both α- and β-esterase bands Est-3 and 4 in digestive juice and Est-2 and 3 in haemolymph were found to be nonspecific. Nonspecific β-esterase band (Est-3) in haemolymph of CB5 (GP) and its syngenic lines withstood a temperature up to 80±1°C for 10 min. No thermostable band was observed in the isozyme zymogram of α-esterase in digestive juice and haemolymph or β-esterase in digestive juice. Overall, this study discusses the presence of esterase heterogeneity in the CB5 (GP) genepool, syngenic lines development, occurrence of specific α- and β-esterase bands in digestive juice and haemolymph and thermostable β-esterase band Est-3 in haemolymph in tropical silkworm Bombyx mori L.     

Enhanced expression of the β II subspecies of protein kinase C in differentiating erythroleukemia cells

Polyclonal antipeptide antibodies which recognize selected isozymes (α, β I, β II, and γ) of the protein kinase C family were used to identify specific subspecies in undifferentiated Friend erythroleukemia cells and in cells triggered to differentiate with hexamethylene bisacetamide. The β II isozyme of protein kinase C was the primary isozyme expressed and its abundance was significantly increased (P < 0.05) in differentiated cells. Differences in immunostaining between control and experimental groups were objectively quantitated by determining percentage transmission of light through cells based on color threshold rather than gray intensity levels. Staining was localized to the cytoplasm predominantly in differentiated cells, whereas nuclei stained more intensely in undifferentiated cells. These results provide immunocytochemical evidence to support the hypothesis that changes in the expression of the β II subspecies of protein kinase C are essential to the programmed maturation of differentiating Friend erythroleukemia cells.     

Affinity labeling of high-affinity α-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). ¹²⁵I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (K_d ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling ¹²⁵I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of M_r ≈ 150 000 revealed as a ¹²⁵I-α-thrombin cross-linked complex of M_r ≈ 180 000. Independent of chemical cross-linking, ¹²⁵I-α-thrombin also formed a covalent complex with a minor, 35 000 M_r, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of M_r 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 M_r binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response.     

Metabolism of 1α-hydroxyvitamin D3 by cytochrome P450scc to biologically active 1α,20-dihydroxyvitamin D3

Cytochrome P450scc (CYP11A1) metabolizes vitamin D3 to 20-hydroxyvitamin D3 as the major product, with subsequent production of dihydroxy and trihydroxy derivatives. The aim of this study was to determine whether cytochrome P450scc could metabolize 1α-hydroxyvitamin D3 and whether products were biologically active. The major product of 1α-hydroxyvitamin D3 metabolism by P450scc was identified by mass spectrometry and NMR as 1α,20-dihydroxyvitamin D3. Mass spectrometry of minor metabolites revealed the production of another dihydroxyvitamin D3 derivative, two trihydroxy-metabolites made via 1α,20-dihydroxyvitamin D3 and a tetrahydroxyvitamin D3 derivative. The K_m for 1α-hydroxyvitamin D3 determined for P450scc incorporated into phospholipid vesicles was 1.4 mol substrate/mol phospholipid, half that observed for vitamin D3. The k_cat was 3.0 mol/min/mol P450scc, 6-fold lower than that for vitamin D3. 1α,20-Dihydroxyvitamin D3 inhibited DNA synthesis by human epidermal HaCaT keratinocytes propagated in culture, in a time- and dose-dependent fashion, with a potency similar to that of 1α,25-dihydroxyvitamin D3. 1α,20-Dihydroxyvitamin D3 (10 μM) enhanced CYP24 mRNA levels in HaCaT keratinocytes but the potency was much lower than that reported for 1α,25-dihydroxyvitamin D3. We conclude that the presence of the 1-hydroxyl group in vitamin D3 does not alter the major site of hydroxylation by P450scc which, as for vitamin D3, is at C20. The major product, 1α,20-dihydroxyvitamin D3, displays biological activity on keratinocytes and therefore might be useful pharmacologically.     

Comparative Biochemistry of a Cytosolic Artiodactyl Glycosidase

Artiodactyls possess abundant neutral glycosidase activity in liver, kidney and intestine. This enzyme is cytosolic and displays a more neutral pH optimum, more acidic isoelectric point and broader substrate range than the corresponding acidic β-galactosidases. The neutral glycosidases were more thermolabile than the respective acidic β-galactosidases and displayed a relative molecular mass approximating 60 kDa. This isozyme appeared to be a minor species in both rat and dog liver. The porcine enzyme was studied in more detail. Porcine neutral glycosidase activity was detected in 45-day gestational fetuses in both liver and kidney but not brain. Fetal kidney activities were about half those observed in adult kidney extracts. Porcine neutral glycosidase was immunologically distinct from acidic β-galactosidase and was immunologically similar to the corresponding isozymes from deer, ovine and bovine liver. Porcine neutral glycosidase was moderately inhibited by d-galactonic acid γ-lactone and strongly inhibited by d-gluconic acid δ-lactone; however, acidic β-galactosidase was not inhibited by the δ-lactone. Inhibition by the γ-lactone was competitive for both enzymes. 4-Methylumbelliferyl-β-d-galactoside, -glucoside and -xyloside competed for the same active site. A polymorphism for fast- and slow-migrating isozymes of porcine neutral glycosidase was observed, which appeared to be under genetic control.     

The system of sulfated galactans from the red seaweed Gymnogongrus torulosus (Phyllophoraceae, Rhodophyta): Location and structural analysis

Sulfated polysaccharides were localized in the cuticle, cortex and medulla of the gametophyte thallus, being more concentrated in the intercellular matrix than in the cell walls. During the water extraction sequence, a small percentage of galactan sulfates (5.1% of dry seaweed) with average low M_r (6–11.4 kDa) were extracted at room temperature without disturbing the cellular arrangement, while sulfated galactans of average medium M_r (18–45 kDa) were obtained by further hot-water extractions (52.4% of dry seaweed), with diorganization of the tissue. The residue (40.0% of dry seaweed) still contained carrageenan-type (major) and agaran-type (minor) galactans. Part of these galactans was extracted with 8.4% LiCl solution in DMSO, from which “pure” κ/ι-carrageenans were isolated.Carrageenans and agarans were extracted in a ratio 1:0.5, showing the highest amount of agaran-structures for a carrageenophyte. The galactans comprise alternating 4-sulfated (major) and non-sulfated (minor) 3-linked β-d-galactopyranose units, and 4-linked α-galactopyranose units with the following substitutions: (i) non-sulfated and 2-sulfated 3,6-anhydro-α-d-galactopyranose residues in the carrageenan-structures, which belong to the κ-family (κ/ι-carrageenans); (ii) 3-sulfated α-l-galactopyranose units and 2-sulfated 3,6-anhydro-α-l-galactopyranose residues in the agaran-structures.Alkaline treatment and alkaline dialysis of the main extracts gave “pure” κ/ι-carrageenans, showing that carrageenan molecules are extracted together with low M_r agarans or agaran-dl-hybrids.     

Characterization of β-endorphin- and α-MSH-related peptides in rat heart

POMC-derived peptides and mRNA have been identified in heart tissue, although POMC processing has not been fully characterized. In the present study, we found that β-lipotropin and ACTH were localized in rat heart, although they were almost entirely converted to β-endorphin- and α-MSH-related peptides. Ion exchange HPLC analysis revealed that β-endorphin(1–31) was further processed to α-N-acetyl-β-endorphin(1–31), which comprised 35.9 ± 0.1% of total immunoreactivity, and smaller amounts of β-endorphin(1–27), β-endorphin(1–26), and their α-N-acetylated derivatives. The predominant α-MSH immunoreactive peptides coeluted with α-MSH and N,O-diacetyl-α-MSH by reverse-phase HPLC, although small amounts of ACTH(1–13)-NH₂ were also present. Thus, multiple forms of β-endorphin and α-MSH are localized in rat heart. β-Endorphin(1–31) is a minor constituent, however, indicating that nonopioid β-endorphin peptides predominate.     

Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1

Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of Kras^G12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV.     

A method for the determination of the pKa of α-n-benzoyl-l-arginine in the presence of an impurity

A method is proposed for the simultaneous detection and determination of minor impurities in weak acid (or bases) along with the evaluation of all relevant pK′s. By use of the method described, the pK′s of α-n-benzoyl-l-arginine and α,ω-dibenzoyl-l-arginine have been accurately determined in the same solution. These values are 3.31 for α-n-benzoyl-l-arginine and 2.24 for α,ω-dibenzoyl-l-arginine. This latter compound was found to exist as a 5.3% impurity in a commercial preparation of α-n-benzoyl-l-arginine.     

Human prostatic steroid 5α-reductase isoforms—A comparative study of selective inhibitors

The present study describes the independent expression of the type 1 and 2 isoforms of human 5α-reductase in the baculovirus-directed insect cell expression system and the selectivity of their inhibition. The catalytic properties and kinetic parameters of the recombinant isozymes were consistent with published data. The type 1 isoform displayed a neutral (range 6–8) pH optimum and the type 2 isoform an acidic (5–6) pH optimum. The type 2 isoform had higher affinity for testosterone than did the type 1 isoform (K_m = 0.5 and 2.9 μM, respectively). Finasteride and turosteride were selective inhibitors of the type 2 isoform (K_i (type 2) = 7.3 and 21.7 nM compared to K_i (type 1) = 108 and 330 nM, respectively). 4-MA and the lipido-sterol extract of Serenoa repens (LSESr) markedly inhibited both isozymes (K_i (type 1) = 8.4 nM and 7.2 μg/ml, respectively; K_i (type 2) = 7.4 nM and 4.9 μg/ml, respectively). The three azasteroids were competitive inhibitors vs substrate, whereas LSESr displayed non-competitive inhibition of the type 1 isozyme and uncompetitive inhibition of the type 2 isozyme. These observations suggest that the lipid component of LSESr might be responsible for its inhibitory effect by modulating the membrane environment of 5α-reductase. Partially purified recombinant 5α-reductase type 1 activity was preserved by the presence of lipids indicating that lipids can exert either stimulatory or inhibitory effects on human 5α-reductase.     

Integrin α5 during early development of Xenopus laevis

The full length sequence of the Xenopus integrin α₅ subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of α₅ mRNA occurs in the embryo. Furthermore, a variant form of the α₅ mRNA is expressed which encodes an integrin α₅ subunit with a truncated cytoplasmic domain. Integrin α₅ mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of α₅ mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the α₅ protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin α₅ subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of α subunit expression in early vertebrate embryos.     

The timecourse of collagen cross-linking

The incorporation of [³H] proline into all major denaturation subunits of acid-extractable skin collagen was followed in vivo over a period of 30 days in young male rats. The radioactivity was incorporated into the α₁ and α₂ subunits at an equal rate. It was then transferred to the β subunits, the ratio of specific activity of α:β becoming unity at about 17 days. No transfer of radioactivity from β to γ collagen was seen during the 30 days of the experiment indicating that β units do not cross-link further to γ during this time. A separate group of rats made lathyritic with β-aminopropionitrile showed a similar rate of collagen cross-linking as reflected by the return of α:β ratios to normal values within about 15 days following the cessation of β-aminopropionitrile administration. In the normal rats radioactive proline was incorporated very rapidly into a fraction of collagen eluted from CM-cellulose with 0.5 M NaCl following the salt gradient. It is proposed that this fraction may represent a fibrogenesis nucleation factor.     

Differences in oligosaccharide pattern of a sample of polar bear colostrum and mid-lactation milk

Although the concentrations of carbohydrate in the colostrum and in the mid-lactation milk of polar bear (Ursus maritimus) were similar, the oligosaccharide patterns differed. The colostrum sample contained Neu5Ac(α2-3)Gal(β1-4)Glc (3′-N-acetylneuraminyllactose), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (A-tetrasaccharide), Fuc(α1-2)Gal(β1-4)Glc (2′-fucosyllactose) and Gal(β1-4)Glc (lactose). The mid-lactation milk contained Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (B-pentasaccharide), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (A-pentasaccharide), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (B-tetrasaccharide), A-tetrasaccharide, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]Glc (3-fucosylisoglobotriose), Gal(α1-3)Gal(β1-4)Glc (isoglobotriose) and lactose. The dominant saccharides in the colostrum were 3′-N-Acetylneuraminyllactose and lactose, whereas isoglobotriose was the dominant saccharide in the mid-lactation milk in which lactose was only a minor component. Isoglobotriose, which had previously been found to be a dominant saccharide in mature milk from the Ezo brown bear, the Japanese black bear and the polar bear, was not found in the polar bear colostrum.     

Two forms of NADP-dependent malic enzyme in expanding maize leaves

Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a K_m (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a K_m (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar K_m (NADP⁺), and the typical increase of K_m for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min^-1 mg protein^-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.Abbreviation MES 2 (N-morpholino)ethane sulfonic acid Work supported by grants from the Consiglio Nazionale delle Ricerche for years 1975 and 1976     

Glass transition temperatures of a ready to eat breakfast cereal formulation and its main components determined by DSC and DMTA

The effect of water content on the glass transition temperatures of a ready to eat cereal formulation was determined, as well as for its major components, oat flour, rice flour and an oat–rice flour blend, in the same ratio as they are present in the formulation. All samples were compression moulded at high temperature and were moisture conditioned in a 10–22% interval (dry basis). Glass transition temperatures (T_g) were measured by differential scanning calorimetry (DSC) and the main mechanical relaxation temperatures (T_α), measured by dynamic mechanical thermal analysis (DMTA). The relaxation temperatures taken at tan δ peaks, were found 20–30 °C larger than T_g. Besides the plasticizing effect of water adequately described by the Gordon–Taylor equation, no differences of T_g (and T_α) values between the major components were obtained at a constant moisture content. The T_g and T_α values of the RTE formulation were found to be about 30 °C lower than its components, a result which was attributed to the plasticizing effect of the minor components in the formulation (sugar and malt extract).     

Chromosomal puff induction in salivary glands from Drosophila hydei by arsenite

During incubation of salivary glands from Drosophila hydei in the presence of arsenite (7.5 × 10⁻⁵ M) the major temperature-sensitive puffs show essentially the same kinetics of development as after anaerobiosis followed by air or during temperature shock. Also the dose-response curve, the labeling intensity after a [³H]uridine pulse and the electronmicroscopic features of the temperature-sensitive chromosome regions are virtually the same during arsenite treatment as those obtained with other treatments. However, one major difference between incubation with arsenite and the other treatments is found in the changes in ATP level. During arsenite treatment the ATP level does not change, whereas during the other treatments the ATP level is reduced. In an attempt to correlate puff-inducing events with changes in the mitochondrial metabolism, the affinity of the α-glycerophosphate oxidase was measured and the related changes in the α-glycerophosphate concentration were studied. During all puff-inducing treatments studied so far, thus also during arsenite treatment, a decrease in affinity of the α-glycerophosphate oxidase and a decrease in α-glycerophosphate concentrations is found in the first 15–20 min of the incubation. Later the concentration of α-glycerophosphate increases again, possibly due to the further decrease in affinity of α-glycerophosphate oxidase.