A comparative study of molluscan and crustacean chitin proteoglycans by carbon-13 NMR spectroscopy. Identification of carbohydrate and amino acid contributions and the determination of amino acid chemical shifts in anhydrous formic acid

• 1.1. ¹³C-NMR spectra of formic acid solutions of chitin proteoglycans from cephalopod pen, lamellibranch siphon sheath and crab cuticle have been determined.
• 2.2. Carbohydrate and amino acid components provide well-defined resonances, completely assignable in the case of hexosamine and partially so for protein amino acids.
• 3.3. The individually unique spectra contain information of compositional and chain environment nature.
• 4.4. Spectral data for each protein amino acid, as a formic acid solution, is presented and compared with values for chemical shifts of amino acids and peptides in water.

     

Modulation of gastric mucosal calcium channel activity by mucus glycoprotein

• 1.1. The effect of gastric mucus glycoprotein on the activity of calcium channel isolated from gastric epithelial cell membrane was investigated. The ⁴⁵Ca²⁺ uptake into the vesicle-reconstituted channels, while only moderately (14%) affected by the intact mucus glycoprotein, was found significantly inhibited (59%) by the acidic glycoprotein fraction. This effect was associated with the sialic acid and sulfate ester groups of the glycoprotein, as their removal caused a loss in the inhibition.
• 2.2. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels showed a 50% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 3.3. The channel protein phosphorylation was inhibited by the acidic mucus glycoprotein, which also interfered with the binding of EGF to the channel protein. The inhibitory effect was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following their removal.
• 4.4. The results suggest that the acidic gastric mucus glycoproteins, by modulating the EGF-controlled calcium channel phosphorylation, play a major role in gastric mucosal calcium homeostasis.

     

Influence of dietary sources of fat on lipid synthesis in mink (Mustela vison) mammary tissue

• 1.1. The fatty acid composition of the triglyceride fraction of mink milk sampled during mid-lactation (day 28 post partum) from two nursing mink was compared to that of plasma samples and to the fatty acid composition of the feed rations used.
• 2.2. Chemical analysis of the triglyceride composition of mink milk demonstrated only minute concentrations of fatty acids with a chain length below C₁₄.
• 3.3. The saturated C_16:0- and C_18:0-unit fatty acids in mink milk made up for 24–40% of the total amount of fatty acids extracted, the remainder being represented by mono and polyunsaturated long-chain (C₁₆-C₂₄) fatty acids.
• 4.4. Preliminary in vitro experiments proved the incorporation of¹⁴C-labelled glucose, acetate or palmitate into triacylglycerols in cultures of mink mammary tissue to be linear for at least 2 hr.
• 5.5. The in vitro capacity for de novo fatty acid synthesis in mink mammary tissue using 14C-labelled glucose or acetate was low, i.e. ranging from 0.096–0.109 nmol/g (fresh tissue)/min, and amounted to only about 5% of that obtained in the case of [¹⁴C]palmitic acid incubation.
• 6.6. Following ¹⁴C-labeIled acetic or palmitic acid incubation of mink mammary tissue neither desaturation nor chain elongation was observed.
• 7.7. In response to long-term feeding on rations with two different sources of animal fat (F = fish oil or L = lard) the influence of compositional changes in dietary neutral lipids on the fatty acid composition of the lipids of mink milk is discussed.

     

Palmitic acid metabolism in hepatopancreas of the freshwater shrimp Macrobrachium borellii

• 1.1. The palmitic acid fate as substrate for the synthesis of either glycerides or other fatty acids was studied in vivo and in the microsomal fraction from hepatopancreas of Macrobrachium borellii.
• 2.2. Most of the palmitic acid administered in vivo circulated to the hepatopancreas, being incorporated mainly in the triacylglycerol (TG) fraction.
• 3.3. Palmitic acid transformations into palmitoleic, stearic and oleic acids were observed in the hepatopancreas.
• 4.4. The in vitro biosynthesis of TG in hepatopancreas was more active than in other tissues. In the microsomal fraction, palmitic acid was also incorporated mainly in TG, and followed the α-glycerophosphate pathway.

     

Neutral amino acid transport in an androgen-dependent epithelium

• 1.1. Transport of neutral amino acids by the isolated seminal vesicle epithelium of normal and gonadectomized guinea pigs has been investigated by measurement of the uptake of 2-amino[1-¹⁴C]-isobutyric acid and 2-methylamino[1-¹⁴C]isobutyric acid.
• 2.2. The V_max for Na-dependent and -independent transport of both amino acids was reduced by gonadectomy but the general transport characteristics appeared to be unchanged by this treatment.
• 3.3. The most likely explanation of the decreased transport is the loss of transporter molecules associated with the tissue regression that follows rapidly on gonadectomy.

     

Sulphhydryl protease inhibitors from pineapple plant stem

• 1.1. Pineapple stem extract, consisting of a mixture of the protease bromelain and sulphhydryl protease inhibitors, was fractionated by gel permeation chromatography.
• 2.2. Inhibitor-containing fractions were further resolved by ion exchange chromatography on DEAE-cellulose, giving 12 chromatographically distinct inhibitory fractions.
• 3.3. These 12 inhibitory fractions all show an inhibition specificity towards bromelain.
• 4.4. Reduction, S-carboxymethylation and refractionation of each of these inhibitory fractions gave, for each fraction, two separated peptides of ca 13 and 40 amino acids in length, respectively.
• 5.5. The amino acid compositions and the N-terminal sequences of these peptides show the inhibitors to be a closely homologous set. Both the constituent peptides of each fraction are microheterogeneous. Each DEAE-cellulose chromatogram peak contains a co-eluting set of iso-inhibitors.
• 6.6. Structural microvariations within these isoinhibitors have a minor influence on inhibitor activity towards bromelain.

     

Amino acid synthesis during hyperosmotic stress in penaeus aztecus postlarvae

• 1.1. Glycine, proline, and taurine are the quantitatively most important amino acid osmolytes in Penaeus aztecus postlarvae.
• 2.2. Taurine dominates the amino acid pool in low salinity, while proline dominates the amino acid pool at higher salinities.
• 3.3. Although not major contributors to the pool, glutamate and alanine are constitutively synthesized from [¹⁴C]glucose and [¹⁴C]glutamate under constant salinity and under hyperosmotic stress treatments.
• 4.4. Proline synthesis from [¹⁴C]-precursors is apparent under constant high (but not low) salinity and is significantly induced by hyperosmotic stress.
• 5.5. No appreciable glycine synthesis was observed from [¹⁴C]glucose or [¹⁴C]glutamate under any experimental conditions.

     

Structural features of carbohydrate chains in human salivary mucins

• 1.1. The structure of carbohydrate chains in the low and high molecular weight mucus glycoprotein forms from submandibular-sublingual saliva of individuals with blood group B was investigated.
• 2.2. Alkaline borohydride reductive cleavage of the glycoproteins yielded in each case a population of neutral (55%) and acidic (45%) oligosaccharide alditols ranging in size from 3 to 16 sugar units.
• 3.3. The predominant neutral oligosaccharides in both glycoprotein forms consisted of 16 and 15 sugar units arranged in triantennary fashion, and carried blood group B and I antigenic determinants.
• 4.4. Three of the oligosaccharides in each glycoprotein contained sialic acid and ranged in size from 3 to 12 sugar units. In two oligosaccharides sialic acid was linked to C3 of galactose and in one to C6 of N-acetylgalactosamine. The sulfated oligosaccharide in both glycoproteins was identified as a pentasaccharide with the sulfate ester group at C6 of N-acetylglucosamine.
• 5.5. The results demonstrate that contrary to the earlier view the low and high molecular weight mucus glycoprotein forms of human saliva contain identical carbohydrate chains.

     

Proteolysis post mortem in North Atlantic krill

• 1.1. The autoproteolytic processes in selected species of North Atlantic krill, Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer) and T. raschii (M. Sars) have been examined at 0°C by following the release of peptides and free amino acids.
• 2.2. The krill contains high levels of peptide hydrolases, and autoproteolysis seems to be due mainly to digestive enzymes localized in the hepatopancreas and the intestinal tract of the animals.
• 3.3. During autoproteolysis the individual amino acids were generally released at rates corresponding to their proportion in the bulk protein of the krill. The major exceptions were alanine which accumulated in amounts larger than was to be expected from the composition of the krill protein, and glutamic acid/glutamine, aspartic acid/asparagine, arginine, and to some extent glycine, proline and serine, which accumulated to a lesser extent than was to be expected.
• 4.4. Storage of krill for 1 week resulted in only minor changes in the total content of amino acids as determined after acid hydrolysis, with the exception of alanine which increased in concentration.
• 5.5. The results suggest that the formation of free alanine is partly due to reactions other than proteolysis.
• 6.6. The release of free amino acids was accompanied by a considerable increase in the amount of small peptides, and glutamic acid/glutamine, aspartic acid/asparagine, glycine and proline tended to accumulate in these peptides.
• 7.7. The autoproteolytic activity of the Thysanoessa species showed seasonal variations, probably in response to food availability. In the case M. norvegica, the results suggest that there are smaller fluctuations in the level of proteolytic enzymes, probably indicating less pronounced variations in the food intake over the year.

     

Isolation and partial amino acid sequence of A 78 kDa porcine gastrin-binding protein

• 1.1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle¹⁵]-gastrin; 17.
• 2.2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides.
• 3.3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55–61). The remaining 7 peptides were derived from the gb-subunit of the gastric H⁺/K⁺-ATPase.
• 4.4. The gastrin-binding activity remained in association with p78, and could be separated from the P-subunit of the gastric H⁺K⁺-ATPase, during chromatography on tomato lectin-Sepharose.
• 5.5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.

     

Driving forces for the uphill transport of amino acids into epidermal brush border membrane vesicles of the sea anemone,Anemonia sulcata (Cnidaria,anthozoa)

• 1.1. The transport of amino acids into membrane vesicles prepared from epidermal tentacle tissue of the sea anemone, Anemonia sulcata, depends on an electrochemical potential difference caused, e.g. by sodium chloride gradients.
• 2.2. Potassium or choline chloride gradients energized the transport less effectively than sodium chloride gradients. Both Na⁺-ions and Cl⁻-ions were required for the amino acid transport.
• 3.3. The uphill transport of amino acids along the downhill movement of driver ions (sodium chloride gradient conditions) was characterized by an overshoot; under sodium chloride equilibrium conditions, however, an accumulation of amino acids within the vesicles could not be measured.
• 4.4. Potassium diffusion potentials in combination with valinomycin indicated that hyperpolarization (vesicle inside negative) and hypopolarization (vesicle inside positive) enhanced or depressed the accumulation of amino acids within the vesicles.
• 5.5. Being at the phylogenetic base of the Eumetazoa, cnidarians show characteristics for the transmembrane transport of amino acids comparable to those established for vertebrates.

     

Control of mucin molecular forms expression by salivary protease: Differences with caries

• 1.1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion.
• 2.2. The protease exhibited optimum activity at pH 7.0–7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, α₁antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases.
• 3.3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects.
• 4.4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.

     

Chemical feeding stimulants for the herbivorous fish,Tilapia zillii

• 1.1. Chemical feeding stimulants for an herbivorous fish, Tilapia zillii have been determined by fractionation and bioassay of substances derived from a model food plant.
• 2.2. Stimulation was produced by amino acids; glutamic acid, aspartic acid, serine, lysine and alanine produced the bulk of stimulatory activity.
• 3.3. These amino acids are among the most abundant in the test plant, and are markedly different from the amino acids found to stimulate feeding in carnivorous fish.
• 4.4. On the basis of these results, a chemically-mediated mechanism of feeding niche separation is postulated.

     

A halogenated amino acid-containing sperm activating peptide and its related peptides isolated from the egg jelly of sea urchins,Tripneustes gratilla,Pseudoboletia maculata,Strongylocentrotus nudus,Echinometra mathaei and Heterocentrotus mammillatus

• 1.1. Twenty-eight peptides were isolated from the egg jelly of sea urchins, Tripneustes gratilla, Pseudoboletia maculata, Strongylocentrotus nudus, Echinometra mathaei (type A and B) and Heterocentrotus mammillatus and their amino acid sequences were determined.
• 2.2. Two of the peptides obtained from T. gratilla egg jelly possessed a bromophenylalanine (Br-Phe) residue in their sequences (Gly-(Br-Phe)-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly and Gly-(Br-Phe)-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly).
• 3.3. All of the peptides elevated cyclic GMP concentrations in the spermatozoa of the respective sea urchin and caused a shift in the apparent mol. wt of a major sperm protein of the respective sea urchin.
• 4.4. They stimulated respiration rates of the spermatozoa of Hemicentrotus pulcherrimus as well as their own species.
• 5.5. One-half maximal concentrations of the peptides for respiratory stimulation of H. pulcherrimus spermatozoa were between 10⁻¹¹ M and 10⁻⁹ M except a methionine-containing peptide which was about 10⁻⁷ M.

     

Protein digestion and ion concentrations in rainbow trout (Salmo gairdnerii Rich.) digestive tract in sea- and fresh water

• 1.1. Rainbow trout maintained in fresh water or Actapted to sea-water for 24 hr were fed casein-based dry diet. After feeding, fish were kept in fresh water (FW) or transferred to artificial sea-water (SW) and sacrificed after 10 or 20 hr.
• 2.2. The digestive tract was separated into five parts: stomach, pyloric caeca region, middle intestine and two equal lengths of rectum.
• 3.3. The content of these parts was analysed for ions Na⁺, K⁺, Cl⁻, Mg²⁺ and for free, peptide and total amino acids.
• 4.4. In the fish stomach all ions, with the exception of Ca²⁺, indicate drinking of sea-water. In the pyloric caeca region Na⁺ appears to be efficiently absorbed in SW fish but influxed in FW fish. In the rectum of SW fish K⁺ appears to be reabsorbed but Na⁺ concentrated in faeces.
• 5.5. Free amino acid concentrations were always higher in gut lumen of SW than in FW fish in respect to time after feeding and portion of intestinal content. Free amino acids constitute at most 7.4–8.7% of total amino acids in the content of pyloric caeca region.
• 6.6. Peptide amino acids, being mostly di-, tri- and tetra-peptides, increased in stomach content from 14.7 to 28.4% of the total, from 6 to 10 hr after a meal in SW fish. Peptide amino acids constituted 80.3–89.0% of total amino acids in intestinal content of the pyloric caeca region. These peptide portions decreased in the mid-intestine (47.5–52.5%) and increased again in the rectum (73.6–76.0%).
• 7.7. It was concluded that in rainbow trout fed in both sea- or fresh water, ion concentrations do not seem to interfere with protein digestion and nutrient absorption in alimentary tract.

     

Metabolic utilization of leucine in a fat and a lean line of chicken

• 1.1. In vivo incorporation into body lipids and breast muscle proteins from l-[U-¹⁴C]leucine was studied in genetically lean or fat male chickens, fed or starved, 1 or 24 hr after intraperitoneal injection.
• 2.2. Lipogensis and portein synthesis from labelled leucine were significantly higher in fat chickens than in lean birds, particularly in those in the fed state.
• 3.3. Radioactivity in the free amino acid pool was greater in fat birds irrespective of the nutritional state.
• 4.4. However, utilization of injected l-[U-¹⁴C]leucine for lipogenesis was no more than 2%.

     

Thermochemistry of N-C bonds hydrolysis in amides,peptides, N-acetyl amino acids and high-energy N-C bonds hydrolysis in N-acetyl imidazole and urea

• 1.1. The reaction enthalpies of hydrolysis of amides, peptides and N-acetyl amino acids were calculated for both ionized and un-ionized forms of reaction components.
• 2.2. The average values of reaction enthalpies of amides, peptides and N-acetyl amino acids hydrolysis were essentially different from each other for ionized forms of reaction components and were equal for un-ionized forms of reaction components in the error interval.
• 3.3. As an example of high-energy N-C bonds N-acetyl imidazol and urea were discussed. It was found that the reaction enthalpies of hydrolysis of above compounds were different from analogous thermodynamic values of hydrolysis of amides, peptides and N-acetyl amino acids for any forms of components.

     

Fatty acid profile of the digestive gland and mantle in the bivalve Macoma balthica

• 1.1. Digestive gland and mantle fatty acids were studied in spring and summer in the bivalve Macoma balthica off the southern coast of Finland. The presence of lipids was also examined histochemically in various clam tissues.
• 2.2. the neutral lipid content of the digestive gland increased ca 4.5-fold during the annual growth period.
• 3.3. Neutral lipid fatty acids of the digestive gland, of which palmitoleic, eicosapentaenoic and palmitic acids were predominant, were clearly distinguished from phospho- and glycolipid fatty acids.
• 4.4. The degree of unsaturation of phospholipid fatty acids was higher in the cold season both in the digestive gland and mantle, mainly due to the titer of eicosapentaenoic acid.

     

Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein

Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues.     

Chemical composition of North Atlantic krill

• 1.1. The main chemical components of Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer) and T. raschii (M. Sars) have been examined.
• 2.2. Protein accounted for 42–47% of the dry weight of M. norvegica and 32–50% of the dry weight of the Thysanoessa species. On a wet weight basis, the protein content was relatively constant and independent of season.
• 3.3. The dominating amino acids in the bulk protein of the krill were glutamic acid/glutamine, aspartic acid/asparagine, glycine, alanine, lysine and leucine.
• 4.4. Lipids were present in amounts of 13–29% of the dry weight in M. norvegica, 15–50% in T. inermis and 12–44% in T. raschii, and the lipid content varied with season.
• 5.5. The main nitrogen extractives in krill, expressed on a dry weight basis, were free amino acids (5–10%), trimethylamine oxide (about 4%), peptides (about 1%) and nucleotides (0.4–1.3%). Trimethylamine and ammonia were present in very low concentrations in living krill.
• 6.6. The amino acids taurine, glycine, proline, arginine, sarcosine and alanine made up 89–93 mol% of the free amino acid pool.
• 7.7. The ash content of krill was in the order of 10–13% of the dry weight, and fluoride represented 1040 and 3200 ppm in the Thysanoessa species and M. norvegioca, respectively.