Collagen diversity in the sea urchin,strongylocentrotus purpuratus

• 1.1. Pepsin insensitive fragments of collalgen extracted from tube feet and peristome have α 1 and α 2 bands that differ in apparent molecular weights from each other and from human type 1 collagen.
• 2.2. A monoclonal antibody that reacts with the ξ I band and a low molecular weight fragment of tube foot collagen does not react with either peristome collagen or human type I collagen.
• 3.3. Measurements of the axial periodicity of native fibers of tube foot and peristone collagens indicate they have D values that differ significantly from each other and from reported values of vertebrate type I collagen.
• 4.4. We propose that there are diverse and specialized types of collagen in sea urchins that are heterogeneously distributed in the extracellular matrix of different tissue.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

Collagenaceous,thiol-containing proteins of cnidarian nematocysts: A comparison of the chemistry and protein distribution patterns in two types of cnidae

• 1.1. Nematocyst structural proteins (NSP) from the sea anemones Aiptasia pallida and Metridium senile and the siphonophore Physalia physalis are primarily low molecular weight collagens linked by disulfide bonds.
• 2.2. NSP patterns resolved by SDS-PAGE revealed a common, major collagen species (40 kDa) in each nematocyst type, together with other collagens and non-thiol-containing proteins.
• 3.3. For each cnidarian, NSP glycosylation profiles were significantly different.
• 4.4. Monoclonal antibodies against Aiptasia NSP demonstrated a differential distribution between capsule wall and thread.
• 5.5. NSP differences would account for the diversity of morphologic and functional types.

     

Two acid phosphatases in sea urchin eggs and embryos

• 1.1. Two types of acid phosphatases from sea urchin eggs and embryos were studied in three Japanese species, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus.
• 2.2. Acid phosphatase type 1, designated AcP-1, hydrolysed only flavin mononucleotide besides p-nitrophenylphosphate. The activity of AcP-1 was not inhibited by NaF and tartrate. This enzyme showed molecular weight between 14,000 and 16,000 by gel filtration through Sephadex G-75.
• 3.3. The higher molecular weight type of acid phosphatase, designated AcP-2, showed relatively high substrate specificity toward ADP and ATP. Molecular weight of AcP-2 ranged from 42,000 to 48,000 by gel filtration through Sephadex G-100.
• 4.4. Some properties of AcP-2 from Sphaerechinus granularis embryos are also described.

     

Protein differences in cilia of mechanoreceptor hair and gill cells of the scallop Mizuhopecten yessoensis

• 1.1. In search for mechanosensory molecules the composition of the ciliary proteins of mechanoreceptor hair cells of the abdominal organ and less mechanosensitive gill cells were compared electrophoretically.
• 2.2. The hair cells and gill cilia were very similar in their polypeptide sets but differed by contents of three axonemal polypeptides with molecular weights of 125, 149 and 300 kilodaltons (kDa) and one membrane polypeptide of 159 kDa.
• 3.3. The membrane polypeptide with a molecular weight of 159 kDa represented approximately 3% of the total ciliary protein of hair cells. There was only a trace of this polypeptide in gill cilia and their membrane fraction.
• 4.4. A peculiarity of ciliary membranes of the hair cells was a high content of the 159 kDa-polypeptide, which constituted more than 20% of the total protein of membrane fraction.

     

Oxygen binding characteristics of Artemia hemoglobin domains

• 1.1. The extracellular hemoglobins of the crustacean Artemia can be split into structural and functional domains by limited proteolysis.
• 2.2. The oxygen affinity of the multi-domain fragments increases linearly with decreasing molecular weight.
• 3.3. Cooperativity is expressed only in the intact dimeric molecule and not at the subunit or multi-domain level.

     

Evidence for two isoforms of carbonic anhydrase II in the leech (Hirudo medicinalis) central nervous system

• 1.1. We dissected, homogenized and prepared ganglia and connectives from the central nervous system of medicinal leeches for SDS gel electrophoresis. The isolated proteins were transferred to nitrocellulose and incubated with affinity column-purified antibodies.
• 2.2. The immunoblots showed a strong positive reaction of a bovine carbonic anhydrase standard at a molecular weight of 29 kDa, and a distinct double-bond at the same molecular weight in the analyzed material.
• 3.3. We demonstrated with rat antibodies that carbonic anhydrase II is detectable in the leech central nervous system as the main isoenzyme.
• 4.4. The biochemical knowledge of carbonic anhydrase reported here agrees well with the immunocytochemical locations, thus affirming the validity of specific staining.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Ca2+-binding and protein-protein interaction domains of the sea urchin extraembryonic coat protein hyalin

• 1.1. As reported previously (Hopper and Robinson, 1990; Int. J. Biochem. 22, 1165–1170) the sea urchin extraembryonic coat protein hyalin undergoes a Ca²⁺-induced self-association into an insoluble gel (gelation) in the presence of Mg²⁺ and/or NaCl.
• 2.2. A 275 kDa peptide fragment, generated by limited tryptic digestion of hyalin, binds Ca²⁺+ but does not undergo gelation in the presence of Ca²⁺, Mg²⁺ and NaCl.
• 3.3. Comparisons between the capacities of hyalin and the 275 kDa peptide fragment to bind Ca²⁺ indicate that the latter binds 88% less Ca²⁺ than hyalin.
• 4.4. However, the presence of Ca²⁺ alone, at a concentration of 5 mM, protects the 275 kDa peptide fragment from further digestion by trypsin mimicking the effect of this cation in protecting hyalin.
• 5.5. Gel exclusion Chromatographie analyses of the 275 kDa peptide fragment, both in the presence and absence of 5 mM Ca²⁺, indicate that this cation does induce self-association of the fragment.
• 6.6. These results provide information on the organization of the functional domains on hyalin which are required for gel formation.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

Characteristics of chromatin release during digestion of nuclei with micrococcal nuclease: Preferential solubilization of nascent rna at low enzyme concentration

• 1.1. Extensive digestion of nuclei with micrococcal nuclease (MNase), commonly used in the analysis of chromatin structure, results in the production of mono- and dinucleosomal chromatin fragments.
• 2.2. Digestion of nuclei from a range of cell types with low enzyme concentrations solubilized high molecular weight polynucleosomal fragments, some ⪢ 22 kb long.
• 3.3. Such digestion conditions also resulted in extensive solubilization of nascent RNA which contributed considerably to the nucleic acid content of the soluble fraction.
• 4.4. We conclude that the contribution of RNA to total nucleic acid content of the soluble fraction should be taken into consideration when nuclei are digested with low concentrations of MNase.

     

Purification and some properties of a Mg2+-activated acid phosphatase from rat testis

• 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
• 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
• 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
• 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
• 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.

     

In vitro Ca deposition by rat matrix vesicles: Is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

• 1.1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media.
• 2.2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment.
• 3.3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment.
• 4.4. The specific activity of the released ALP was at least 5-fold higher than the residual activity.
• 5.5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or βGP-dependent calcium deposition.
• 6.6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

     

Haemolytic activity in the brown shrimp (Penaeus californiensis holmes) haemolymph

• 1.1. Natural haemolytic activity in brown shrimp (Penaeus californiensis) haemolymph was detected using mouse erythrocytes as target cells. This activity is unrelated to agglutinating and phenoloxidase activity, but it is another probable component of the shrimp defence system.
• 2.2. The haemolytic reaction is time and dose dependent, and a serine-protease is involved.
• 3.3. The haemolytic factor is thermolabile and has an apparent molecular weight of 23.5 kDa.

     

A possible cause of heterogeneity of mammalian apurinic/apyrimidinic endonuclease

• 1.1. Mammalian major apurinic/apyrimidinic (AP) endonuclease, APEX nuclease (M_r 35.4 kDa) was purified from HeLa cells. A hybrid protein (M_r 36.4 kDa), which was expressed in BW2001 strain cells of E. coli, comprising human APEX nuclease headed by 10 additional amino acids was also purified.
• 2.2. The purified preparations were frequently associated with 31-, 33- and 35-kDa peptides having AP endonuclease activity.
• 3.3. The 33- and 35-kDa peptides were suggested to be formed from the hybrid protein or APEX nuclease during their purification processes by proteolytic cleavage with subtilisin-like protease. The 31-kDa peptide was thought to be produced by chemical cleavage of the aspartyl-prolyl bond of APEX nuclease.
• 4.4. The results support the notion that some of AP endonuclease heterogeneity based on the molecular weight difference are caused by proteolytic (and chemical) cleavage of a species of AP endonucleases during the extraction and purification.

     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Modification of membrane protein organization during in vitro aging of human erythrocytes

• 1.1. In in vitro aged human erythrocytes, the presence of protein clusters can be found on the membrane; these clusters are made up of peptides held together by disulfide bridges, since they can be nearly completely dissociated by dithiothreitol treatment.
• 2.2. SDS-polyacrylamide gel electrophoresis after dithiothreitol dissociation indicates that the aggregates are made of peptide fragments with a molecular weight ranging from 20 to ~ 110 kdalton; none of these fragments correspond to an intact protein component of the membrane.
• 3.3. Their formation results from oxidation and proteolysis of membrane, and perhaps cytoplasmic proteins.

     

Isolation and partial characterization of vitellin from the egg of the giant tiger prawn,penaeus monodon

• 1.1. The results obtained from the present study show that vitellin (yolk protein), a glycolipoprotein derived from the giant tiger prawn, Penaeus monodon, consists of four polypeptides, Ep1, Ep2, Ep3 and Ep4, with molecular weights of 168, 104, 83 and 74 kDa, respectively. This protein was purified using SDS-PAGE and gel-elution.
• 2.2. Antisera against Ep2, Ep3 and Ep4 fractions showed very strong specific binding reactivity when reacted with shrimp ovaries at developing stages.
• 3.3. No crossreaction was observed among Ep2, Ep3 and Ep4 fractions of vitellin. However, anti-Ep2 and anti-Ep3 were demonstrated to be able to react with Ep1 fraction. The homogenates obtained from ripe ovaries and hemolymph of vitllogenesis females showed an immuno-identical pattern to those obtained from egg extracts.
• 4.4. The proteolysis mapping for each fraction showed that Ep1, the high molecular weight fraction, contained Ep2 and Ep3 fragments. These results suggest that Ep1 may be a precursor for Ep2 and Ep3.