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.

     

Partial characterization of the hemoglobin from Hubbs' beaked whale (Mesoplodon carlhubbsi)

• 1.1. Blood samples were obtained from an adult female Hubbs' beaked whale and her fetus.
• 2.2. Two major hemoglobins were demonstrated by cellulose acetate electrophoresis and were purified by ion exchange chromatography from each specimen.
• 3.3. The relative amounts of these components were different in the adult and fetus.
• 4.4. Both of these hemoglobins have a higher affinity for oxygen than normal human hemoglobin.
• 5.5. Maternal and fetal hemoglobins were separated and the N-terminal amino acid of each of these hemoglobins was found to be valine.
• 6.6. Tryptic peptide separation and amino acid analyses of the purified polypeptide chains indicated that the hemoglobins of the fetal sample were identical to those of the maternal.

     

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.

     

Fluxes of dissolved amino acids between sea water and Echinaster

• 1.1. Measurement of free amino acid (primary amine) influx and efflux into the starfish, Echinaster, were accomplished utilizing improved methods of sea water purification and analysis.
• 2.2. Specimens placed in amino acid depleted sea water (5 × 10⁻⁸ M) demonstrated net release as measured with the fluorescamine method. Similarly, specimens placed in the same water to which amino acid mixtures had been reintroduced to normal levels demonstrated net uptake.
• 3.3. A mathematical model indicated an equilibrium amino acid concentration (when influx equals efflux) of 5.26 × 10⁻⁷ M, or about one fourth the level of natural sea water.
• 4.4. Since at normal environmental levels (20.65 × 10⁻⁷ M) net flux is inward by a ratio of nearly 4-1, it is concluded that the previous suggestions of some workers that such would not be the case for marine invertebrates are no longer valid.
• 5.5. The net uptake of amino acid from environmental levels would account for 5.67% of the measured total respiration if all were being metabolized.
• 6.6. This figure appears to be in line with the previously developed hypothesis that the epidermis largely obtains its nutrition directly from the environment. However, the real benefit of the uptake mechanism may be to prevent loss of the body amino acid pools.

     

Isolation,characterization, and comparison of hemolytic peptides in nematocyst venoms of two species of jellyfish (Chrysaora quinquecirrha and Cyanea capillata)

• 1.1. Hemolysins of the sea nettle, Chrysaora quinquecirrha, and the lions' mane jellyfish, Cyanea capillata, collected in the Delaware Bay were partially purified by sequential gel-filtration and high performance liquid chromatography.
• 2.2. The nematocyst contents of both jellyfish had hemolytically active fractions containing large quantities of glycine and serine along with an unknown amino acid residue.
• 3.3. The Chrysaora hemolysin appeared to have a mol. wt greater than 6000 but less than 10,000 daltons.
• 4.4. Glycophorins were the most effective inhibitors to the hemolysins at the lowest concentration.

     

Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum

• 1.1. Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000–400,000 with a peak at about MW 200,000.
• 2.2. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000–600,000 (77% of the fraction NSILA or 28% of total serum NSILA).
• 3.3. Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000–200,000 and 35,000–100,000 fractions and corresponded to 37% of total serum NSILA.
• 4.4. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000–100,000.
• 5.5. Low MW NSILA (<15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000–600,000. This complex was not bound by Con-A Sepharose.

     

Free amino acids in fluids and tissues of Mytilus galloprovincialis in relation to the environment. Their behaviour as an index of normality of metabolism

• 1.1. A population of Mytilus galloprovincialis has been sampled at 6 different stations of the Bay of Naples (Italy) to analyse the behaviour of free amino acids (FAA) of proteic or non-proteic nature, and the relative parameters of environment.
• 2.2. Thirty FAA of proteic and non-proteic nature have been determined in deproteinized tissue and fluids of M. galloprovincialis.
• 3.3. The most frequent FAA were taurine, as representative of the non-proteic amino acid, cystine, alanine and glutamic acid for non-essential proteic amino acids; valine for the essential proteic amino acids; and other ninhydrin-positive constituents.
• 4.4. There were differences in the environment of the colonies of M. galloprovincialis among the several stations and substantial differences in the macromorphological aspect of the animal were found in relation to their source.
• 5.5. The authors conclude that the FAA content is connected essentially to the regulation of the osmotic pressure and that their concentration represents an index of normality of the metabolism (INM): at FAA concentration total values higher than 550 μmole/g of dry tissue corresponds a negative INM with habitat of osmotic stress, while to concentration values between 0 and 550 μmole/g of dry tissue INM is positive or normal with vital habitat for M. galloprovincialis.
• 6.6. The shape of the concentration curves of the single free amino acids was analysed station by station.

     

Rat brain aryl acylamidase: Further characterization of multiple forms

• 1.1. Two fractions of aryl acylamidase (EC 3.5.1.13) were further separated from rat brain extracts at pH 7.5 by ammonium sulfate precipitation and Bio-Gel chromatography.
• 2.2. 1,2,3,4-Tetrahydro-β-carboline competitively inhibited (67%) fraction-1 but slightly inhibited (13%) fraction-2. Tetrahydroharman, 6-hydroxy-tetrahydroharman and harminic acid slightly inhibited both fractions. Harmalol inhibited fraction-1 but enhanced fraction-2. 6-Methoxy-harman, 6-methoxy-harmalan and harmaline enhanced both fractions.
• 3.3. Pargyline did not affect either fraction. Methiothepin, cyproheptadine and chlorimipramine inhibited fraction-1 but stimulated fraction-2.
• 4.4. Neostigmine moderately (30%) inhibited AAA-2 but did not have any significant effect on AAA-1.
• 5.5. These results indicate that the β-carboline compounds might play a role in regulating activity of AAA-1 and 2 in brain.
• 6.6. Both fractions might be related to serotonergic neurons but only AAA-2 might be associated with acetylcholinesterase.

     

Phosphorylation of a 25,000-dalton protein in slow and fast muscles of the chicken

• 1.1. Protein phosphorylation in intact chicken latissimus dorsi muscle, slow anterior (ALD) and fast posterior (PLD), was compared.
• 2.2. A major difference in [³²P]phosphate incorporation was found between the ALD and PLD in a 25,000-dalton heat soluble protein.
• 3.3. The 25,000-dalton protein was purified from both the ALD and PLD.
• 4.4. The two proteins had similar amino acid composition and both contained approximately 1 mole phosphate per mole of protein.
• 5.5. The difference in their content of radioactive phosphate was determined to be due to faster turnover in the ALD.

     

The effect of heparin on the porcine lymphocyte chromatin—I. The comparative study of DNA in different chromatin fractions

• 1.1. Porcine lymphocyte chromatin in the solution of 0.15 M NaCl + 0.01 M Tris. pH 7 treated with heparin liberated 30% protein and 7.5% DNA to the supernatant.
• 2.2. DNA from the supernatant and the pellet fractions as well as from control chromatin were isolated in identical conditions.
• 3.3. No significant changes were observed in spectral properties and melting points in SSC of comparable DNA specimens.
• 4.4. It was noted, however, that DNA of the supernatant is subject to denaturation in the process of isolation, which, apart from the difference in protein composition of the supernatant and the pellet fractions, suggests different chromatin structure of these fractions.

     

On the turnover of tissue lipids in the mouse during dietary deprivation

• 1.1. The double isotope ratios of individual lipid fractions in major tissues of mice were determined before and after a period of total dietary deprivation.
• 2.2. Several significant alterations in these ratios were caused by this treatment, with a marked individuality evident in the response of separate tissues.
• 3.3. The increased metabolic flux overall was contributed to by increased rates of lipid degradation in liver, and active mobilization from the peripheral depots.
• 4.4. Significant utilization of phospholipid fractions for energy purposes was also noticeable, however. along with tissue specific variation in the patterns of both double isotope ratios and specific radioactivities.
• 5.5. These data are discussed in relation to the comparative priorities of synthesis and degradation in the different tissue and lipid sources in response to this physiological perturbation.

     

The lysozyme of the plaice Pleuronectes platessa L.

Lysozyme from the serum of the plaice, Pleuronectes platessa L., has been purified 78-fold with chitin coated cellulose.

• 1.2. Further purification on CM-cellulose yielded a single band on acrylamide electrophoresis, exhibiting lysozyme activity.
• 2.3. The quantitative amino acid composition of plaice serum lysozyme is reported.
• 3.4. The mol. wt is identical with hen egg white lysozyme.
• 4.5. A method is described for identifying fractions with lysozyme activity in polyacrylamide gels.

     

Biochemical variations in three species of prairie dogs (Cynomys)

• 1.1. Blood proteins were studied by polyacrylamide disc gel electrophoresis in three species of prairie dogs, Cynomys gunnisoni, C. leucurus, and C. ludovicianus.
• 2.2. The sera were separated into 13–15 fractions and the three species could be distinguished by both qualitative and quantitative differences in their serum patterns.
• 3.3. Qualitatively, variations in the occurrence and number of slow albumin fractions are diagnostic at the species leel.
• 4.4. Quantitative differences were most apparent in variation in the mobility of the major albumin fraction and the transferrin fraction.

     

The influence of seasonal changes on energy metabolism in Mytilus edulise (L.).—III. Anaerobic energy metabolism

• 1.1. Seasonal changes in the accumulation of end products after 48 hr of exposure to air and in the composition of the free amino acid pool were studied in Mytilus edulis.
• 2.2. The accumulation levels of succinate and acetate showed only weak seasonal changes.
• 3.3. Conversion of succinate to propionate was high in summer and virtually zero in winter
• 4.4. Alanine and most other free amino acids were present in relatively high concentrations in summer and early autumn and reached minimal values in winter and early spring.
• 5.5. Exceptions were glutamate, aspartate and taurine, which showed hardly an season related changes and glycine, which changed inversely to the majority of the free amino acids.
• 6.6. The anaerobic formation of alanine was inversely proportional to the endogenous concentration.
• 7.7. The only other free amino acids affected by anaerobiosis were glutamate and aspartate, which respectively increased and decreased under these conditions.

     

Anaerobic metabolism in sublittoral living Mytilus galloprovincialis in the mediterranean—IV. Role of amino acids in adaptation to low salinities during anaerobiosis and aerobiosis

• 1.1. When Mytilus galloprovincialis were transferred from 38 to 19%. sea water (S), the metabolism became anaerobic for at least 8 hr. After 24 hr the animals were entirely aerobic again.
• 2.2. Upon transfer to 19%. S, the total free amino acid concentration in haemolymph doubled within 4 hr, remaining nearly constant thereafter, up to 48 hr.
• 3.3. In the posterior adductor muscle a strong decrease of alanine and glycine occurred at 48 hr exposure to 19%. S, and a smaller decrease of glutamate; taurine remained relatively constant. When transferred again to 38%. S after 14 days, a strong overcompensation occurred in the concentrations of alanine and proline, and a smaller overcompensation in those of threonine and serine.
• 4.4. In the gill no distinct change in the amino acid pool occurred during 14 days of exposure, with the exception of a decrease in serine. When transferred again to 38%. S, a strong overcompensation occurred in alanine, proline, glycine and serine, and a smaller in glutamate and threonine.
• 5.5. No evidence for anaerobic metabolism in the decrease of the amino acid pool was found.
• 6.6. M. galloprovincialis is less able to adapt to low salinities than the more euryhaline M. edulis.

     

Uptake of haemolymph glycoprotein by the fat body of Calpodes ethlius (lepidoptera). Evidence that the mechanism does not involve desialylation

• 1.1. Haemolymph and fat body of Calpodes ethlius larvae were examined for the presence of sialic acid.
• 2.2. Haemolymph contained low molecular weight substances that absorbed maximally at 549 nm after reaction with periodic acid and thiobarbituric acid (Warren assay).
• 3.3. Ion exchange chromatography of haemolymph ultrafiltrate gave two chromogenic fractions. The principal chromogen failed to give a direct Ehrlich or resorcinol test for sialic acid.
• 4.4. The thin layer chromatographic mobilities of the chromogens differed from those of N-acetylneuraminic acid.
• 5.5. No sialic acid was released by enzymatic or acid hydrolysis of haemolymph or fat body proteins.

     

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.

     

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.

     

Biosynthesis of membrane and a membrane glycoprotein in Tetrahymena pyriformis

• 1.1. A membrane glycoprotein was isolated from Tetrahymena surface membrane with properties similar to mammalian erythrocyte glycoprotein.
• 2.2. This membrane glycoprotein had a specific acitivity less than whole membrane at various time intervals up to 40 hr.
• 3.3. In double labelling experiments, glucosamine contributed a greater degree of the labelling of this glycoprotein than did amino acids.
• 4.4. Greater incremental increases in the glucosamine radioactivity over amino acids at the longer time intervals suggests that the carbohydrate and protein moieties may have independent metabolic pathways.
• 5.5. When Tetrahymena was grown in proteose peptone-tryptone in the presence of labelled amino acid, the specific activity of the surface membrane was less than one-tenth of the specific activity obtained in defined growth media. Under these same conditions, there were only slight changes in the specific activities of glucosamine in the membrane.
• 6.6. Data is presented suggesting independent synthesis of the protein and oligosaccharide from different pools.