Incorporation of intravenously injected (2-(14)C) acetate into tissue lipids of hamsters during the last hour of 3-, 6-, and 24-hour periods of hypothermia.

The in vivo incorporation of total lipid ¹⁴C from [2-¹⁴C]acetate is decreased in kidney, liver, and small intestine tissue from 3-, 6-, and 24-hr hypothermic hamsters compared to tissues from normothermic animals. The length of time in hypothermia affects hamster tissues differently; thus, ¹⁴C activity: decreases with time in kidney; increases with time in liver; and increases at 3 and 6 hr but decreases from 6 to 24 hr of hypothermia in small intestine.Tissues from hypothermic hamsters incorporated a greater percentage of [2-¹⁴C] acetate into free sterols and diglycerides and a smaller percentage into phospholipid than did corresponding tissues from normothermic hamsters.The percentage of total fatty acid ¹⁴C activity found as polyunsaturated fatty acid ¹⁴C activity increases in hypothermic kidney, liver, and small intestine with a decrease in the percentage of ¹⁴C activity measured in the saturated fatty acids. Esterification of fatty acid was inhibited in all tissues taken from hypothermic hamsters.     

Trypsin activity in the small intestine of rats after application of copper and zinc

In vivo experiments with Sprague-Dawley rats were conducted in order to explore the influence of Cu²⁺, Zn²⁺ as well as of the combinations of both on the activity of trypsin. The solutions of the trace elements were given per os, the animals were killed 30 min after the applications, and the activity of trypsin was determined in the juice of the small intestine by usingN ^α-benzoyl-L-arginine-p-nitroanilide (L-BAPA) as the substrate. The activity of trypsin depends on the concentration of the trace elements. When Cu²⁺ ions are applied, there is a minimum activity at 10⁻⁵ mol Cu²⁺/L and a maximum at 10⁻⁴ mol Cu²⁺/L. When giving Zn²⁺ ions, a minimum of trypsin activity is found at 10⁻⁵ mol Zn²⁺/L and a maximum at 5×10⁻⁶ mol Zn²⁺/L. On the whole, the trypsin activity is lower when the Cu²⁺/Zn²⁺ combinations are applied compared to the addition of the single trace elements. On principle, a good conformity of the in vivo results was found with in vitro results.     

Metabolic Fate of Sarcosyl-14C-glycine in Normal Young Rats

The metabolic fate of the non-physiological synthetic dipeptide, sarcosyl-[2-¹⁴C]glycine, was investigated in normal young rats in vivo and in vitro compared to that of seryl-[2-¹⁴C]glycine as a typical example of common physiological dipeptides. The radioactive dipeptides were synthesized from sarcosine or L-serine and [2-¹⁴C]glycine in our laboratory. When radioactive sarcosylglycine was given intraperitoneally, about 30% of the dose was excreted in the urine, and more than 90% of the urinary radioactivity was present in the sarcosylglycine fraction. The recovery of radioactivity in the the expired carbon dioxide and body protein was 8 and 50% of the dose, respectively, during a 24-h period. When the labeled serylglycine was given, the recovery of radioactivity in the urine was 8% of the dose, and 15% in the expired carbon dioxide. In slices of the kidney, liver and small intestine from normal rats, serylglycine was rapidly and almost completely hydrolyzed, and a large amount of free glycine was released. However, sarcosylglycine was hardly hydrolyzed to the corresponding amino acids in the liver and small intestine, and only slightly in the kidney. These results suggest that a considerable amount of sarcosylglycine given intraperitoneally was rapidly excreted into the urine of normal young rats, reflecting less sensitivity to hydrolysis by tissue peptidase(s) when compared to serylglycine.     

The effect of replacing lactose by starch on protein and fat digestion in milk-fed veal calves

Replacing dairy components from milk replacer (MR) with vegetable products has been previously associated with decreased protein and fat digestibility in milk-fed calves resulting in lower live weight gain. In this experiment, the major carbohydrate source in MR, lactose, was partly replaced with gelatinized corn starch (GCS) to determine the effect on protein and fat digestibility in milk-fed calves. In total, 16 male Holstein-Friesian calves received either MR with lactose as the carbohydrate source (control) or 18% GCS at the expense of lactose. In the adaptation period, calves were exposed to an increasing dose of GCS for 14 weeks. The indigestible marker cobalt ethylenediaminetetraacetic acid was incorporated into the MR for calculating apparent nutrient digestibility, whereas a pulse dose of chromium (Cr) chloride was fed with the last MR meal 4 h before slaughter as an indicator of passage rates. The calves were anesthetized and exsanguinated at 30 weeks of age. The small intestine was divided in three; small intestine 1 and 2 (SI1 and SI2, respectively) and the terminal ileum (last ~100 cm of small intestine) and samples of digesta were collected. Small intestinal digesta was analysed for α-amylase, lipase and trypsin activity. Digestibility of protein was determined for SI1, SI2, ileum and total tract, whereas digestibility of fat was determined for SI1, SI2 and total tract. Apparent protein digestibility in the small intestine did not differ between treatments but was higher in control calves at total tract level. Apparent crude fat digestibility tended to be increased in SI1 and SI2 for GCS calves, but no difference was found at total tract level. Activity of α-amylase in SI2 and lipase in both SI1 and SI2 was higher in GCS calves. Activity of trypsin tended to be higher in control calves and was higher in SI1 compared with SI2. A lower recovery of Cr in SI2 and a higher recovery of Cr in the large intestine suggest an increased rate of passage for GCS calves. Including 18% of GCS in a milk replacer at the expense of lactose increased passage rate and decreased apparent total tract protein digestibility. In the small intestine, protein digestion did not decrease when feeding GCS and fat digestion even tended to increase. Overall, effects on digestion might be levelled when partially replacing lactose with GCS, because starch digestion is lower than that of lactose but fat digestion may be slightly increased when feeding GCS.     

Increased diet viscosity by oat β-glucans decreases the passage rate of liquids in the stomach and affects digesta physicochemical properties in growing pigs

Rheological properties of digesta play a role in digesta passage kinetics through the gastrointestinal tract, in turn affecting nutrient absorption kinetics. Therefore, we studied the effects of diet viscosity on digesta passage and physicochemical properties in pigs. Twenty male growing pigs (35 kg body weight at the start) were assigned to one of five diets with increasing dietary concentrations of β-glucans (BG; from 0 % to 10 %), in exchange for maize starch. After a 17-day adaptation period, pigs were euthanised and the mean retention time (MRT) of digesta solids (TiO₂) and liquids (Cr-EDTA) in the stomach, and proximal and distal half of the small intestine was quantified. In the stomach, the MRT of liquids, but not of solids, increased when dietary BG level increased (6 min per % dietary BG, P = 0.008 and R² = 0.35). Concomitantly, stomach DM content (5 g/kg per % dietary BG, P < 0.001 and R² = 0.53) and apparent digesta viscosity (56 Pa × s at 1/s shear rate per % dietary BG, P = 0.003 and R² = 0.41) decreased. In the proximal half of the small intestine, no effects of dietary BG level were observed. In the distal half of the small intestine, water-binding capacity (WBC) of digesta increased (0.11 g/g digesta DM per % dietary BG, P = 0.028 and R² = 0.24) and starch digestibility decreased (0.3% per % dietary BG, P = 0.034 and R² = 0.23) when dietary BG level increased. In the colon, apparent digesta viscosity at 45/s shear rate increased (0.1 Pa × s per % dietary BG, P = 0.03 and R² = 0.24) in the proximal half of the colon, and digesta WBC increased (0.06 g/g digesta DM per % dietary BG, P = 0.024 and R² = 0.26) in the distal half of the colon when dietary BG level increased. To conclude, increasing dietary BG level caused the MRT of liquids, but not that of solids, to increase in the stomach, resulting in reduced separation of the solid and liquid digesta fractions. This caused dilution of the stomach content and reduction in digesta viscosity when dietary BG levels increased. Effects of dietary BG level on physicochemical properties in the proximal small intestine were absent and may have been due to a low DM content. The WBC of digesta in the distal small intestine and colon increased when dietary BG level increased, as did apparent digesta viscosity in the proximal colon. This likely reflects the concentration of BG in digesta when moving through the gastrointestinal tract.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Neb-TMOF, the trypsin modulating oostatic factor of gray fleshfly Neobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 by D-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X = NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) and D-Asn (XII). The influence of these peptides on trypsin biosynthesis in N. bullata was determined in vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [D-His)⁶]- and [D-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performed in vitro on heart of Tenebrio molitor we found that Neb-TMOF and [Phe(p-NH₂)⁶-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Interaction of Salivary and Midgut Proteins of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> with Soybean Trypsin Inhibitor

Feeding of Helicoverpa armigera larvae on semi-synthetic diet containing Soybean trypsin inhibitor (STI) resulted in disappearance of STI sensitive protease in salivary and midgut protease extract. This might be due to in situ inhibition by dietary STI. STI was largely degraded within 1 h of incubation with total salivary protease (1:1). Degradation was relatively low in midgut proteases. STI interacting proteins were isolated from saliva and midgut extracts of larvae fed on STI supplemented diet using affinity column. Most of the isolated proteins showed caseinolytic activity in zymogram. Denovo sequencing data of seven different peptides selected from trypsin digested total protein showed similarity to chymotrypsinogen, serine protease, aminopeptidase N, peroxidase, hypothetical protein and muscle specific protein.     

Translocation of sitosterol and related compounds in Pelargonium hortorum and helianthus annuus

The translocation of several plant sterols and a triterpene was studied in geranium and sunflower plants. Upward translocation of sitosterol-[¹⁴C] and β-amyrin-[¹⁴C] was shown within 48 hr to the upper parts of a geranium plant sectioned previously above the roots. Downward translocation of sitosterol-[¹⁴C] from the leaf of application was evident in intact plants after 48 hr. In addition to free sitosterol-[¹⁴C] considerable amounts of sitosteryl-[¹⁴C] glycoside and traces of sitosteryl-[^14C] ester were found in most parts examined. Very slow downward translocation of cholesterol-[¹⁴C] but not of desmosterol-[¹⁴C], sitosteryl-[¹⁴C] palmitate or β-amyrin-[¹⁴C] was shown in geranium. In sunflower no downward translocation of cholesterol-[³H], sitosteryl-[³H] acetate or palmitate could be detected. In geranium, sitosteryl-[¹⁴C] glycoside translocated downward from the leaf of application to all other plant parts, except other leaves, and was found in these parts after 10 days as the unchanged glycoside, free sterol and steryl ester. The effect of drying the plant parts on the recovery of radioactive steroidal material is discussed. Traces of a water soluble, dialyzable form of sterol-[¹⁴C] were also detected in dried geranium roots after treatment with strong acid or alkali.     

Digestive Tract Cell Proliferation and Food Consumption Patterns of Ha/Icr Mice

The relationship between the daily pattern of food consumption and the proliferation rate of the qesophagus, stomach, forestomach, small intestine and colon of Ha/ICR mice was examined. Proliferative activity was determined by [³H]TdR incorporation on a wet weight tissue basis, along with selective counting of labelled nuclei. Under conditions of ad libitum feeding with a 12 hr light cycle (lights on at 0600) mice eat most of their food during the dark period. A distinct circadian rhythm was observed in the oesophagus, stomach, forestomach and colon with the peak of [³H]TdR incorporation between 0400 and 0600 and the nadir between 1600 and 1800. Although a circadian fluctuation was observed in the small intestine, its amplitude was much less than in other areas. This rhythmic change in proliferation rate could be phase shifted by allowing the mice to feed only between 0800 and 1600 for 14 days. Under these conditions the peak in proliferative activity occurred between 1800 and 2000. Fasting reduced the daily level of proliferative activity in all of the digestive tract sites studied, and for all areas except the oesophagus greatly reduced or eliminated the circadian fluctuation. the forestomach and colon were the most influenced by fasting with 24 hr [³H]TdR incorporation reduced to 30–40% of the control value. Refeeding following a 48 hr fast produced a rapid increase in proliferative activity peaking at levels well above the control value at 16 hr after the onset of refeeding. the major exception to this was the small intestine which slowly returned to the control value during the first 24 hr. Partial refeeding produced a diminished refeeding response. Once the normal pattern of food consumption was re-established following refeeding the normal proliferative fluctuations were again observed.     

Decreased muscarinic binding sites in small intestine from mice treated with neostigmine

It has been reported by several authors that animals given repeated sublethal doses of an organophosphate, acetylcholinesterase (AChE) inhibitor, develop tolerance to its toxicity. This phenomenon seems to be due, at least partially, to a decrease of central and peripheral cholinergic receptors. In the present study, we report that a decrease of muscarinic receptors, as measured by [³H]-quinuclidinyl benzilate (³H-QNB) binding, occurs in the small intestine of mice treated with the carbamate, AChE inhibitor, neostigmine. Male mice were given neostigmine in the drinking water at daily increasing concentrations (from 20 to 1000 ppm). Methylatropine (20mg/kg, i.p.) was administered twice a day for the same period to two groups of control and neostigmine-treated animals. AChE activity was inhibited 60–70% in small intestine and diaphragm and [³H]-QNB binding was significantly reduced in the small intestine of neostigmine-treated mice; both the number of receptors and the affinity were lower than control. This decrease was not present in the tissue of mice given methylatropine together with neostigmine. Administration of methylatropine alone caused a significant increase of [³H]-QNB binding in the small intestine.     

Insect Trypsin Modulating Oostatic Factor (Neb-TMOF) and Its Analogs: Preliminary Structure/Biological Function Relationship Studies

Summary Neb-TMOF, the trypsin modulating oostatic factor of gray fleshflyNeobellieria bullata, is a hexapeptide with the following sequence: H-Asn-Pro-Thr-Asn-Leu-His-OH. It has been isolated from vitellogenic ovaries in 1994. TMOF, the newly discovered insect peptide, inhibits trypsin biosynthesis in the gut, lowers yolk polypeptide concentration in the hemolymph and strongly inhibits ecdysone biosynthesis by larval ring glands. It is interesting that this short non-protected peptide contains in its molecule two Asn residues at positions 1 and 4 and His at its C-terminus. To obtain information about the role of the His-6 and Asn-4 residues we synthesised two series of Neb-TMOF analogs, modified: (1) in position 6 byd-His (I), His(Bzl) (II) and Phe(p-X) derivatives, where X=NH₂ (III), NO₂ (IV), OEt (V) and OH (VI) and (2) in position 4 by such amino acid residues as Ser (VII), Thr (VIII), Gly (IX), Asp (X), Glu (XI) andd-Asn (XII). The influence of these peptides on trypsin biosynthesis inN. bullata was determinedin vivo. In preliminary investigations, we found that Neb-TMOF, [Phe(NH₂)⁶], and [Phe(NO₂)⁶]-Neb-TMOF inhibited trypsin biosynthesis, whereas [d-His)⁶]- and [d-His(Bzl)⁶]-Neb-TMOF were inactive. In further biological studies performedin vitro on heart ofTenebrio molitor were found that-TMOF and [Phe(p-NH₂)⁶]-Neb-TMOF showed weak cardioexcitatory activity, about 30% of the cardioexcitatory activity of proctolin, an insect neuromodulating peptide.     

Appearance of New Protein Species on the Cell Surface during Sexual Differentiation in the Heterobasidiomycetous Yeast,Tremella mesenterica

The effects of neural blockers on the pancreatic enzyme secretion in response to an intraluminal infusion of soybean trypsin inhibitor and HCl were investigated. The stimulation of pancreatic enzyme secretion upon the intraluminal infusion of soybean trypsin inhibitor was not blocked by atropine, but was completely blocked by guanethidine. The intraluminal infusion of 0.08 n HCl, which is known as a potent secretagogue of secretin, caused a rapid augmentation of trypsin output, which was not blocked by atropine or guanethidine. Preinjection of CR-1392 (1.5 mg/kg, i.p.), which is a strong cholecystokinin receptor antagonist, completely blocked the pancreatic response to soybean trypsin inhibitor, but not that to 0.08 n HCl. This inferred that guanethidine specifically suppressed the CCK-release from the small intestine.

These findings suggest that the pancreatic enzyme secretion in response to soybean trypsin inhibitor is mainly mediated by CCK, and that adrenergic modulation would be involved in the CCK-mediated pancreatic enzyme secretion in response to soybean trypsin inhibitor.     

山东产野生大豆胰蛋白酶抑制剂的初步研究

该实验建立了HPLC测定大豆胰蛋白酶抑制剂(STI)活性的方法,并对山东产野生大豆(G.soja)与同地区产的黑豆和黄豆(G.max)的胰蛋白酶抑制活性差异进行了比较.用耦合了胰蛋白酶的亲合色谱柱对野生大豆的STI进行分离纯化,紫外分光光度法比较3种大豆的STI含量;PCR结合TA克隆技术对野生大豆STI中的Kunitz型(KSTI)蛋白基因编码区的氨基酸顺序进行初步测定.结果发现,山东产野生大豆的STI活性和含量均高于同地区产的黑豆和黄豆;山东产野生大豆的KSTI蛋白基因编码区的氨基酸顺序与已知的Tia型基本一致,仅第59位氨基酸由于单核苷酸的置换发生了Ser→Thr的转变,此位置位于活性中心附近.研究表明,山东产野生大豆胰蛋白酶抑制活性较强,且含量高.     

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH₂)p. ATP also increased the formation of cyclic [¹⁴C]AMP in enterocytes that had been preincubated with [¹⁴C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I_sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.     

Trade-off between digestion and respiration in two airbreathing callichthyid catfishes Holposternum littorale (Hancock) and Corydoras aeneus (Gill)

In callichthyid catfishes, the posterior intestine is modified to function as an air breathing organ by being air-filled, thin-walled and highly vascularized. These modifications make it unsuitable for digestive functions and digesta has to be transported quickly through this region to minimize disruption of vital respiratory functions. However, the weak muscles of the wall of the respiratory intestine make this problematic. It is hypothesized that the unidirectional ventilatory air current within the respiratory intestine is responsible for the quick transport of digesta through the respiratory intestine. To verify this, movement of digesta through the alimentary tract was examined in Hoplosternum littorale and Corydoras aeneus that were either allowed to breathe air or prevented from air breathing. When air breathing was prevented, digesta was not transported to the rectum in H. littorale and there was a 94% reduction in the amount of digesta in the rectum of C. aeneus. This study suggests that the anterior digestive intestine facilitates the passage of air although it is filled with digesta. The anterior digestive intestine packages digesta into a string of slightly compressed boluses, creating an air channel in the digestive intestine thus allowing air to pass unimpeded.     

Autoradiographic and biochemical evidence for the systemic translocation of systemin in tomato plants

The movement of systemin, the 18-amino-acid polypeptide inducer of proteinase inhibitors in tomato (Lycopersicon esculentum L.) plants, was investigated in young tomato plants following the application of [¹⁴C]systemin to wounds on the surface of leaves. Wholeleaf autoradiographic analyses revealed that [¹⁴C]systemin was distributed throughout the wounded leaf within 30 min, and then during the next several hours was transported to the petiole, to the main stem, and to the upper leaves. The movement of [¹⁴C]systemin was similar to the movement of [¹⁴C]sucrose when applied to leaf wounds, except that sucrose was slightly more mobile than systemin. Analyses of the radioactivity in the petiole phloem exudates at intervals over a 5-h period following the application of [¹⁴C]systemin to a wound demonstrated that intact [¹⁴C]systemin was present in the phloem over the entire time, indicating that the polypeptide was either stable for long periods in the phloem or was being continually loaded into the phloem from the source leaf. The translocation pathway of systemin was also investigated at the cellular level, using light microscopy and autoradiography. Within 15 min after application of [³H]systemin to a wound on a terminal leaflet, it was found distributed throughout the wounded leaf and was primarily concentrated in the xylem and phloem tissues within the leaf veins. After 30 min, the radioactivity was found mainly associated with vascular strands of phloem tissue in the petiole and, at 90 min, label was found in the phloem of the main stem. Altogether, these and previous results support a role for systemin as a systemic wound signal in tomato plants.The authors acknowledge the Washington State University Electron Microscope Center and staff for their technical advice and collaboration. We also thank Greg Wichelns for growing our plants and Dr. Steven Doares for providing [³H]systemin. This research was supported in part by the Washington State College of Agriculture and Home Economics Project No. 1791 and National Science Foundation grants IBN 9117795 and IBN 9104542     

Stimulative Effect of a Casein Hydrolysate on Exocrine Pancreatic Secretion That is Independent of Luminal Trypsin Inhibitory Activity in Rats

We have previously demonstrated that proteins could stimulate pancreatic secretion independently of luminal bile-pancreatic juice (BPJ) in a BPJ-diverted rat. To determine whether luminal protease-independent pancreatic secretion occurs in normal rats with BPJ returned to the upper small intestine, we investigated the pancreatic secretory response to intraduodenal instillation of a casein hydrolysate or the synthetic trypsin inhibitor, FOY 305, at concentrations which could almost equally inhibit hydrolysis of the synthetic substrate for trypsin with the luminal content. FOY 305 at 10 μg/ml and casein hydrolysate solutions at both 100 and 200 mg/ml similarly inhibited approx. 80% of the tryptic activity in the luminal contents of the proximal small intestine. Intraduodenal administration of casein hydrolysate solutions (100 and 200 mg/ml) significantly increased pancreatic secretion in a dose-dependent manner. However, intraduodenal administration of FOY 305 (10 μg/ml) was ineffective for stimulating pancreatic secretion. These results demonstrate that dietary protein enhances pancreatic secretion independently of the masking of luminal trypsin activity in rats.     

Activation of Rice Aminopeptidase by Anions and Some Properties of the Enzyme

In order to determine which proteases are responsible for the autolysis of krill, the effects of several protease inhibitors on the autolysis and protease activities of krill were investigated.

Homogenates of whole bodies, and the cephalothorax and abdomen parts of frozen krill were equilibrated at 37°C at different pHs between 2 to 10 and allowed to stand for 16 hr, following which the increase in the TCA soluble fraction was monitored. ¹⁴C-Hemoglobin (¹⁴C-Hb) hydrolyzing activity was also measured using each homogenate as a crude enzyme preparation. The degree of autolysis and the ¹⁴C-Hb hydrolyzing activity were maximum at pH 5 ~ 8 for the parts studied. The hydrolytic activity was highest in the cephalothorax, followed by that in the whole body and then the abdomen.

The effects of inhibitors on the ¹⁴C-Hb hydrolyzing activity were examined, and it was seen that soybean trypsin inhibitor (STI), diisopropyl fluorophosphate (DFP) and leupeptin significantly inhibited the activity at neutral pH, and pepstatin, monoiodoacetic acid (IAAcid) and leupeptin were effective at acidic pH for all the parts. Investigation of the effects of inhibitors on the autolysis at 20°C at pH 4 and 7 by SDS–polyacrylamide gel electrophoresis indicated that the autolysis of the cephalothorax and whole body at pH 7 was suppressed a little by STI and the autolysis of the abdomen and whole body at pH 4 was significantly inhibited by iodoacetamide (IAA) and leupeptin.

These results suggest that the main proteases responsible for the autolysis of krill are trypsin like-proteases at neutral pH and cathepsins (B, H and L types) at acidic pH.     

Biosynthesis and secretion of tropoelastin by chick aorta cells

Cells were isolated from the aortae of 17-day old chick embryos by digestion of the vessels with a combination of trypsin and collagenase. When these cells were incubated in suspension culture in Krebs-Ringer media containing pancreatic trypsin inhibitor and radioactive amino acids, they synthesized and secreted labeled proteins into the media. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the secreted proteins labeled with [¹⁴C]proline revealed two major components. The larger component with an approximate molecular weight of 125,000 had a [¹⁴C]hydroxyproline content consistent with a form of procollagen. The molecular weight of 70,000 and [¹⁴C]hydroxyproline content of the second component was consistent with that previously reported for tropoelastin extracted from chick aortae. By following the kinetics and secretion of tropoelastin labeled with [³H]valine, we have estimated that 17 minutes are required to synthesize and secrete the molecule under these experimental conditions.