Ca-binding glycoprotein from the spicules of the octocoral Leptogorgia virgulata

• 1.1. A Ca-binding component (CaBC) was isolated from the soluble matrix of spicules of the gorgonian Leptogargia virgulata.
• 2.2. The CaBC is a glycoprotein with apparent mol. wt between 70,000 and 100,000, and contains very high levels of aspartate.
• 3.3. The CaBC probably plays an important role in the formation of the coral spicules.

     

A very stable and potent fibrinolytic enzyme found in earthworm Lumbricus rubellus autolysate

• 1.1. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity.
• 2.2. It also showed a clot-forming activity in the fibrinogen- or plasma-added system.
• 3.3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and 15,000 in the autolysate.
• 4.4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N-terminal amino acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

     

Crystalline style proteins from bivalve molluscs

• 1.1. Proteins from crystalline styles of twelve species of bivalve mollusc were examined under different gel electrophoresis conditions and stained to reveal both protein and carbohydrate.
• 2.2. Native extracts of styles produced relatively few protein bands, however denaturation with SDS resulted in much more complex zymograms.
• 3.3. All species possessed several prominent high mol wt glycoproteins.
• 4.4. Eulamellibranchia all had a major non-glycosylated protein at approx. 62,000 mol. wt.
• 5.5. Most Filibranchia had a major non-glycosylated protein at 37,000–50,000 mol. wt.
• 6.6. Eulamellibranchia were a much more homogeneous group than the Filibranchia.

     

Some physical and chemical properties of purified nematocysts of Hydra attenuata pall. (Hydrozoa,cnidaria)

• 1.1. A method for purifying undischarged nematocysts from Hydra and other cnidarians is described.
• 2.2. Isolated cysts (relative densities 1.22–1.24) evaginate their tubular content even after previous dehydration.
• 3.3. The cyst wall is permeable to dyes of mol. wts up to 600,000.
• 4.4. Approximately two-thirds of the cyst's dry wt are soluble proteins. Eighty per cent of them are of low mol. wt and highly anionic, presumably serving as binding sites for Ca²⁺ and Mg²⁺.
• 5.5. The other 20% includes 30 different proteins amongst them toxins and enzymes (phospholipase and little proteases but no collagenase, chitinase or hyaluronidase).

     

Troponin from nematode: purification and characterization of troponin from Ascaris body wall muscle

• 1.1. A troponin-like protein was isolated from body wall muscle of Ascaris and separated into three components, the mol. wts of which were approx. 58,000, 36,000 and 20,000 respectively.
• 2.2. The three components were designated as troponin-T (TNT), troponin-I (TNI) and troponin-C (TNC) in order of mol. wt, since each component had properties similar to the respective components of vertebrate skeletal-muscle troponin.
• 3.3. Ascaris troponin were localized on actin filaments with a 44 nm repeat, an approximately 4 nm longer repeat than vertebrate troponin.

     

The subunit structure of Daphnia pulex hemoglobin

• 1.1. The extracellular hemoglobin of Daphnia pulex has an apparent molecular weight of 430,000–470,000 by gel chromatography and an S_20,w = 16.9 at pH 7.0.
• 2.2. Purified hemoglobin contains one heme per 18,000–20,000 g protein. The polypeptide chains are heterogeneous with mol. wts between 31,000–37,000. Some high mol. wt (M_r = 53,000–86,000) material is also present.
• 3.3. The hemoglobin dissociates at pH 10.5 in EDTA into 3S material which can be digested with subtilisin into 16,000 mol wt heme-containing polypeptide chains.
• 4.4. The amino acid composition of the intact hemoglobin is identical to that of the heme-containing fragments generated by proteolytic digestion of the 3S material.
• 5.5. These results are consistent with the hypothesis that D. pulex hemoglobin is composed of subunits containig two heme groups per 35,000 mol. wt polypeptide chain.

     

Characterization of proteins from mitochondrial ribosomes of Locusta migratoria by three different two-dimensional gel electrophoretic methods and comparison with cytoplasmic ribosomal proteins

• 1.1. Proteins were isolated from subunits of mitochondrial and cytoplasmic ribosomes of Locusta migratoria and were analyzed by means of two-dimensional gel electrophoreses using three different electrophoresis systems.
• 2.2. Using the system of Czempiel et al. (1976) proteins from whole locust mitochondrial ribosomes (combined subunits) were separated into 72 spots; proteins from the large and small subunits resulted in 48 and 29 spots respectively.
• 3.3. The mol. wt distribution of mitochondrial ribosome proteins was estimated by using the electrophoresis system of O'Farrell (1975). These mol. wts are in the range of 11,000–56,000, the average mol. wt is about 29,500. Assuming one copy of protein per ribosome this gives a total mol. wt for the protein part of mitochondrial ribosomes of ca. 2.1 x 10⁶.
• 4.4. Parallel separation of cytoplasmic and mitochondrial ribosome proteins was achieved using the system of Geyl et al. (1981). Cytoplasmic ribosome proteins produced 65 spots and revealed a more alkaline character than mitochondrial ribosome proteins.

     

Trypsin inhibitors in serum of adult and suckling rats and in rat milk

• 1.1. Trypsin inhibitors in serum from adult and suckling rats and in rat milk were studied by gel filtration and electrophoresis in casein-agarose gels. In addition trypsin binding properties and inhibiting activity in the presence of low mol. wt substrates were studied.
• 2.2. In adult rat serum 6 trypsin inhibitors were found, most of these having not been described before. One inhibitor, di-macroglobulin is a homologue of human α₂-macroglobulin. Two inhibitors in the α₁- and α₂-regions of the electropherogram had a mol. wt of about 200,000. The third group of inhibitors was eluted together with albumin and had electrophoretic mobilities in the di-α₂- and β-region.
• 3.3. In the serum of the newborn rat the inhibitors of the third protein peak dominated, especially that in the α₂-region. In later developmental stages the inhibitor pattern became increasingly similar to that of the adult. A specific inhibitor, α₂-acute phase globulin, was found in the neonatal rat but disappeared in later developmental stages.
• 4.4. The trypsin inhibitors in rat milk were dominated by inhibitors in the third protein peak after gel nitration and had the same electrophoretic mobilities as the inhibitors of the corresponding fraction in serum. No low mol. wt trypsin inhibitors specific for milk were found.
• 5.5. Rat milk trypsin inhibiting activity seems to have a higher resistance to low pH-values than the inhibiting activity of rat serum.
• 6.6. The trypsin inhibitor pattern in the serum of the suckling rat is similar to that of milk, indicating an absorption of inhibitors from milk. The milk inhibitors may have the function of preventing protein breakdown in the intestine of the suckling rat.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Comparative study of three different forms of both native and subunit vitellogenin identified in quail plasma

• 1.1. Three different species of native vitellogenin, designated Vgα, Vgβ and Vgγ, were detected by gradient polyacrylamide gel electrophoresis (2–16%) in the plasma of untreated mature female quail and in the plasma of estrogen-induced female or male quail. The molecular weights of Vgγ, Vgβ and Vgα (in order of increasing size) were estimated to be 400,000 to 450,000 (by PAGE) or 466,000 (by analytical ultracentrifugation).
• 2.2. DEAE-cellulose chromatography resolved vitellogenin-containing plasma into peak IV, fractions of which contained Vgα and Vgβ in equal quantities, and peak III, fractions of which contained Vgα, Vgβ and Vgγ in varying proportions.
• 3.3. Peak IV fractions disssociated to give two bands (designated Vg1 and Vg2) on SDS-polyacrylamide gel electrophoresis. Pooled peak III fractions and plasma from untreated female or estrogen-induced female and male quail dissociated to give three bands (Vg1, Vg2 and Vg3). The mol. wt of Vg1, Vg2 and Vg3 were approx. 232,500, 212,000 and 194,000, respectively.
• 4.4. Peak III and peak IV vitellogenin fractions were shown to have similar amino acid compositions except that the peak III vitellogenin fraction contained twice as much cystine as the peak IV vitellogenin fraction (2.2 vs 1.1 mol%). The peak IV vitellogenin fraction contained more serine than the peak III vitellogenin fraction (11.8 vs 10.9 mol%) and more phosphorus (0.584 vs 0.516 nmol/μg protein).
• 5.5. Vgα or Vg1, in trace amounts, were detected in the plasma of untreated male quail.
• 6.6. The amino acid contents, phosphorus contents, and mol. wt of quail vitellogenins were similar to published values for other egg-laying species.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

Differential expression of salivary mucin bacterial aggregating activity with caries status

• 1.1. The low and high mol. wt mucin forms were isolated from saliva of caries-resistant (CR) and caries-susceptible (CS) individuals, and assessed for their bacterial aggregating potential towards S. mutans and S. sanguis, the common cariogenic microorganisms encountered in the oral cavity.
• 2.2. The high mol. wt mucin from both groups of subjects exhibited similar protein and carbohydrate content, but the level ofcovalently bound fatty acids was significantly lower in the CR group. The mucin from CR group showed only a weak inhibitory potential, and no inhibitory activity was observed with the mucin of CS group.
• 3.3. The low mol. wt mucins from both groups, while displaying compositional similarities, showed a marked variation in the bacterial aggregating activity. With both bacteria, the activity of the mucin from CR group was at least 128-fold greater than that of CS group.
• 4.4. The conversion of the high mol. wt mucin to a low mol. wt form through the action of salivary protease produced in both groups enhancement in mucin's bacterial aggregating capacity. This enhancement was, however, considerably less pronounced in the case of mucin from CS group.
• 5.5. The results for the first time demonstrate that the bacterial aggregating epitope of salivary mucins is expressed to a greater extent in CR individuals, and that this epitope is apparently more accessible to bacteria in the low mol. wt mucin form.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Nuclear distribution pattern of tumour-associated nonhistone protein of mol. wt 48,000.

• 1.1. As a further step toward characterizing nonhistone protein of mol. wt 48,000 which was found to be much more abundant in animal tumour cells than in normal ones [Krajewska W.M., Lipinska A., Marszatek M., Kiliańska Z., Wojtkowiak Z. and Kłyszejko-Stefanowicz L. Cell. Biochem. Funct. 8, 79–89 (1990)] its intranuclear localization in hamster liver and Kirkman-Robbins hepatoma was studied. The protein was identified by immunoblotting technique in the presence of antibodies against polypeptide of mol. wt about 48,000 from Kirkman-Robbins hepatoma.
• 2.2. Distribution of antigen with mol. wt of 48,000 in nuclear fractions representing different levels of nuclear material organization i.e. in nucleoli, nuclease-sensitive and nuclease-resistant fractions, and extensive nuclease digestion products separated by size on Bio-Gel A-50m, implied the structural role of this component.
• 3.3. Fractionation of endogenously digested nuclei into low salt extract, high salt extract and nuclear matrix revealed that in normal liver the antigen studied is associated with nuclear matrix while in hepatoma this component appeared in high salt extract.
• 4.4. These results suggest that polypeptide with mol. wt of 48,000 is a shuttling protein which may be involved in reorganization of nuclear matrix during neoplastic transformation.

     

Creatinine amidohydrolase: Some properties of the partially purified and purified enzyme

• 1.1. Creatinine amidohydrolase from Pseudomonas sp. has a pH optimum of 8.0 and is activated by divalent metals manganese, magnesium, zinc and cobalt.
• 2.2. It is acid labile but shows good stability at 55°C in alkaline solutions.
• 3.3. It has a mol. wt in the region of 248,000 and Michaelis constants of 31.7mM and 80 mM for creatinine and creatine respectively.
• 4.4. Results indicate that the enzyme molecule contains 8 subunits of similar mol. wt.

     

Apparent and real variability in the presence and metal contents of metallothioneins in the crab Carcinus maenas including the effects of isolation procedure and metal induction

• 1.1. Reducing conditions must be maintained throughout the procedure of isolating metallothioneins from crabs. Dithiothreitol is preferred to 2-mercaptoethanol for long-term protection.
• 2.2. Two metallothioneins (10,100 and 4100 mol. wt, respectively) in the hepatopancreas of the crab Carcinus maenas showed great variability between individual crabs as to their presence and to their contents of Zn, Cu and Cd.
• 3.3. The 10,100 mol. wt metallothionein was induced in the laboratory by exposure to Cu and Cd, and variably by Zn-exposure. Laboratory induction did not raise significantly the total metal content of 0.88 ± 1.13 g atoms/mol protein of this metallothionein in crabs from the Firth of Clyde, Scotland.
• 4.4. The 4100 mol. wt metallothionein was not induced in the laboratory by exposure to Cu, Cd or Zn. This metallothionein in crabs from the Firth of Clyde, Scotland, contained 0.27 ± 0.34 g atoms of total Cu, Cd and Zn per mole of protein.

     

Identification of proteolytic isozymes from Antarctic krill (Euphausia superba) in an enzymatic debrider

• 1.1. A partially purified krill extract (enzymatic debrider) intended for clinical use was electrophoretically characterized by polyacrylamide gel electrophoresis (PAGE) and by crossed immunoelectrophoresis (CIE) using polyclonal rabbit antibodies.
• 2.2. Three main types of proteolytic enzymes (serine proteinase, carboxypeptidase A and B) with mol. wts of 33,000, 28,000 and 35,000, respectively, could be separated by SDS—g-PAGE under reducing conditions.
• 3.3. Routine CIE analysis of krill samples revealed four protease-active immunoprecipitates. Two of these precipitates were associated with the proteinase activity, one with carboxypeptidase A and one with carboxypeptidase B.
• 4.4. Improving resolution of CIE by extending electrophoresis in the first dimension permitted separation of three serine proteinases of which two were isozymes (II and III) and the third one was unique (I).
• 5.5. Furthermore carboxypeptidase A could also be separated into two isozymes (AI and AII) while carboxypeptidase B still exhibited one single component.
• 6.6. Six individual immunoprecipitates were thus identified and proved to be related to the protease activity. Highly purified enzymes were used as references in CIE and tandem-CIE to establish identification of each enzyme in the krill mixture.

     

Ribosome inactivating protein-like activity in seeds of diverse Cucurbitaceae plants

• 1.1. Seed extracts of 20 plants species belonging to the family Cucurbitaceae were examined for their ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Eleven extracts were found to inhibit protein synthesis by about or over 90%, seven extracts produced about 80% inhibition, one caused about 70% inhibition and one brought about approx. 40% inhibition, when the extracts were tested at a final concentration of 10 μg per ml.
• 3.3. All of the seed extracts possessed potent mid-term abortifacient activity.
• 4.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the seed extracts disclosed the existence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000 Da. This band probably accounts for the protein synthesis inhibiting and mid-term abortifacient activities.
• 5.5. There was a similarity in the electrophoretograms of seed extracts of plants belonging to the same genus.

     

Investigation of ribosome inactivating protein-like activity in tissues of cucurbitaceae plants

• 1.1. Extracts of roots, seeds and fruits of seventeen plant species belonging to Family Cucurbitaceae were examined for the ability to inhibit protein synthesis in rabbit reticulocyte lysate and induce mid-term abortion in mice.
• 2.2. Out of the 22 tissue extracts examined, 16 were found to inhibit protein synthesis by >90%, three caused 65–85% inhibition and 3 caused <25% inhibition.
• 3.3. In general, there was a close correlation between protein synthesis inhibiting activity and mid-term abortifacient activity of the tissue extracts.
• 4.4. SDS-PAGE of the tissue extracts revealed the presence of a Coomassie Blue-stainable band with a mol. wt of ca 30,000. The data suggest that this band is responsible for the protein synthesis inhibiting and mid-term abortifacient activities.

     

Characteristics of the in vitro assembly of brain tubulin of Cyprinus carpio

• 1.1. Tubulin has been isolated from brain of carp (Cyprinus carpio), acclimated to summer temperatures (16–20°C), and its in vitro reassembly behavior has been characterized.
• 2.2. Among the striking properties of this tubulin preparation is the temperature profile showing a high level of polymerization at the environmental temperature of carp.
• 3.3. The critical tubulin concentration for assembly was 0.8 mg/ml, which was higher than mammalian tubulin purified by the cycle procedure.
• 4.4. The microtubular protein showed three high mol.wt component and a minor component of about 43,000 daltons was also found to copurify with tubulin.