Response to environmental salinity of Na-KATPase activity in individual gills of the euryhaline crab Cyrtograpsus angulatus

The occurrence, localization and response to environmental salinity changes of Na⁺-K⁺ATPase activity were studied in each of the individual gills 4-8 of the euryhaline crab Cyrtograpsus angulatus from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Na⁺-K⁺ATPase activity appeared to be differentially sensitive to environmental salinity among gills. Upon an abrupt change to low salinity, a differential response of Na⁺-K⁺ATPase activity occurred in each individual gill which could suggest a differential role of this enzyme in ion transport process in the different gills of C. angulatus. With the exception of gill 8, a short-term increase of Na⁺-K⁺ATPase specific activity was observed in posterior gills, which is similar to adaptative variations of this activity described in other euryhaline crabs. However, and conversely to that described in other hyperregulating crabs, the highest increase of activity occurred in anterior gills 4 by 1 day after the change to dilute media which could suggest also a role for these gills in ion transport processes in C. angulatus. The fact that variations of Na⁺-K⁺ATPase activity in anterior and posterior gills were concomitant with the transition to hyperregulation indicate that this enzyme could be a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab. The results suggest a differential participation of branchial Na⁺-K⁺ATPase activity in ionoregulatory mechanisms of C. angulatus. The possible existence of functional differences as well as distinct regulation mechanisms operating in individual gills is discussed.     

Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages

Tissue-specific age-dependent changes were observed in Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na⁺K⁺- and Ca²⁺-ATPase in testes and only Ca²⁺-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na⁺K⁺-, Ca²⁺- and Mg²⁺-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.     

Responses of Na+-K+ ATPase activities from dog olfactory tissue to selected odorants.

Na⁺-K⁺ ATPase activity from nerve ending particle (NEP) fractions of dog olfactory tissue homogenates showed different patterns of response to odorants. Similar turbinal groupings were removed from the right and left sides of the septum in the nasal cavity and NEP preparations were tested with eight different odor compounds, including 2-keto alkane homologs and the optical isomers d- and l-carvone. Odorant stimulation of Na⁺-K⁺ ATPase activity from paired turbinal groupings did not show bilateral symmetry. Different patterns of stimulation were observed for each turbinal grouping and for each odorant. A stimulation of over 200% was observed in one preparation in response to 2-nonanone.A study of the response of Na⁺-K⁺ ATPase activity from individual turbinals showed that the enzyme in each turbinal had a different response pattern to six different odorants. Inhibitory and stimulatory responses were observed for the individual turbinal NEP preparations. These results support the proposal that odor sensing initiation may occur through odorant perturbation of the Na⁺-K⁺ ATPase activity.     

FRACTIONATION OF OLFACTORY TISSUE HOMOGENATES. ISOLATION OF A CONCENTRATED PLASMA MEMBRANE FRACTION

Abstract— Differential and sucrose-density-gradient centrifugation techniques were used for studies on the separation of subcellular particles from rabbit brain and olfactory tissue. Comparisons were made among various fractions from the two types of tissue. These comparisons included protein concentration and enzyme activities of the individual fractions as well as their distribution in subfractions from density gradient separations. In tissue whole homogenates, the percentage of total ATPase activity as ouabain sensitive Na⁺-K⁺ ATPase activity was about 4 times greater in brain cortex (63 per cent) than in olfactory tissue (17 per cent). Cytochrome oxidase and Na⁺-K⁺ ATPase activities were used to indicate the presence and the concentration of mitochondria and of the plasma membranes. A fraction with properties similar to the mitochondria plus nerve ending fraction from brain homogenates (fraction B) was obtained from olfactory tissue. Nerve ending concentration subfractions (B₂) were prepared from the B primary fractions. Plasma membrane subfractions were obtained by osmotic shock treatment of B₂, In the fraction of plasma membrane from olfactory tissue (E₂), 56 per cent of the total ATPase activity was Na⁺-K⁺ ATPase activity. In E₂ from brain 71 per cent was Na⁺-K⁺ ATPase activity. Deoxycholate (DOC)-treated fractions containing nerve endings from brain preparations showed much greater increase in cytochrome oxidase activity than did similar fractions from olfactory tissue. DOC treatment increased the NADH cytochrome c reductase activity of all fractions and subfractions from brain, while it decreased activity in all but one fraction from olfactory tissue. DOC treatment decreased both the Mg²⁺ and Na⁺-K⁺ ATPase activities in both types of tissue. Electron photomicrographs of olfactory B₂, B₃, E₂ and E₃ show clear morphological differences among these subfractions. The presence of possible cilia and basal bodies on vesicles in B₂ gives morphological evidence for the presence of terminal swellings in this subtraction in agreement with enzyme marker activity results.     

EFFECT OF UNILATERAL VISUAL DEPRIVATION AND VISUAL STIMULATION ON THE ACTIVITIES OF ALKALINE PHOSPHATASE, ACID PHOSPHATASE, Na+−K+ ACTIVATED Mg2+ CATALYSED ADENOSINE TRIPHOSPHATASE AND ON THE CONTENT OF SODIUM AND POTASSIUM IONS OF THE OPTIC LOBE OF ADULT PIGEON

—A study was made of the effects of unilateral visual deprivation and stimulation upon the activities of alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2), Na⁺-K⁺ activated Mg²⁺ catalysed ATPase (EC 3.6.1.4) and upon the Na⁺ and K⁺ contents of the optic lobe of adult pigeon (Columba livia). Visual deprivation was achieved by eyelid suturing or by enucleation and maintained for 1–9 weeks. Unilateral visual stimulation was maintained for 75 min following 72 h of darkness. A statistically significant increase in the activity of alkaline phosphatase activity was observed in the optic lobe after unilateral visual deprivation whereas unilateral visual stimulation resulted in the opposite effect. Acid phosphatase activity was found to be unchanged under all experimental conditions. Na⁺-K⁺ ATPase activity was found to increase significantly following unilateral visual stimulation and following eyelid suturing in the corresponding optic lobes; unilateral enucleation resulted in a decrease in the Na⁺-K⁺ ATPase activity. An increase in the enzyme activity was found to be associated with an increase in the level of Na⁺-ion and a decrease in the level of K⁺-ion, and vice versa.     

Actein inhibits the Na+-K+-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells

The Na⁺-K⁺-ATPase is a known target of cardiac glycosides such as digitoxin and ouabain. We determined that the enzyme also is a target of the structurally-related triterpene glycoside actein, present in the herb black cohosh. Actein’s inhibition of Na⁺-K⁺-ATPase activity was less potent than that of digitoxin, but actein potentiated digitoxin’s inhibitory effect on Na⁺-K⁺-ATPase activity and MDA-MB-453 breast cancer cell growth. We observed different degrees of signal amplification for the two compounds. Actein’s inhibitory effect on ATPase activity was amplified 2-fold for cell growth inhibition, whereas digitoxin’s signal was amplified 20-fold. Actein induced a biphasic response in proteins downstream of ATPase: low dose and short duration of treatment upregulated NF-κB promoter activity, p-ERK, p-Akt and cyclin D1 protein levels, whereas higher doses and longer exposure inhibited these activities. Actein and digitoxin may be a useful synergistic combination for cancer chemoprevention and/or therapy.     

Studies on the glycoprotein component of (Na+-K+)ATPase from guinea pig brain

In this study an attempt was made to elucidate the possible mechanism of the brain microsomal (Na⁺-K⁺)ATPase inhibition based on the assumption that glycoprotein part of the enzyme is exposed on the outer membrane surface. In our experiments the modification with concanavalin A of sugar end groups exposed by neuraminidase treatment resulted in a significant decrease of the brain (Na⁺-K⁺)ATPase activity. The percentage of the enzyme inhibition by concanavalin A binding to the neuraminidase-treated preparation corresponds to the amount of liberated sialic acids. The modification of the glycoprotein part of the brain (Na⁺-K⁺)ATPase complex by neuraminidase and concanavalin A treatments did not affect K⁺-nitrophenylphosphatase activity.     

Cardiac sarcolemmal Na+-Ca2+ exchange and Na+-K+ ATPase activities and gene expression in alloxan-induced diabetes in rats

To determine the sequence of alterations in cardiac sarcolemmal (SL) Na⁺-Ca²⁺ exchange, Na⁺-K⁺ ATPase and Ca²⁺-transport activities during the development of diabetes, rats were made diabetic by an intravenous injection of 65 mg/kg alloxan. SL membranes were prepared from control and experimental hearts 1-12 weeks after induction of diabetes. A separate group of 4 week diabetic animals were injected with insulin (3 U/day) for an additional 4 weeks. Both Na⁺-K⁺ ATPase and Ca²⁺-stimulated ATPase activities were depressed as early as 10 days after alloxan administration; Mg²⁺ ATPase activity was not depressed throughout the experimental periods. Both Na⁺-Ca²⁺ exchange and ATP-dependent Ca²⁺-uptake activities were depressed in diabetic hearts 2 weeks after diabetes induction. These defects in SL Na⁺-K⁺ ATPase and Ca-transport activities were normalized upon treatment of diabetic animals with insulin. Northern blot analysis was employed to compare the relative mRNA abundances of _--subunit of Na⁺-K⁺ ATPase and Na⁺-Ca²⁺ exchanger in diabetic ventricular tissue vs. control samples. At 6 weeks after alloxan administration, a significant depression of the Na⁺-K⁺ ATPase _-- subunit mRNA was noted in diabetic heart. A significant increase in the Na⁺-Ca²⁺ exchanger mRNA abundance was observed at 3 weeks which returned to control by 5 weeks. The results from the alloxan-rat model of diabetes support the view that SL membrane abnormalities in Na⁺-K⁺ ATPase, Na⁺Ca²⁺ exchange and Ca²⁺-pump activities may lead to the occurrence of intracellular Ca²⁺ overload during the development of diabetic cardiomyopathy but these defects may not be the consequence of depressed expression of genes specific for those SL proteins.     

Na+-K+ ATPase and carbonic anhydrase activities in larvae,postlarvae and adults of the shrimp Penaeus japonicus (Decapoda,Penaeidea)

• 1.1. Activities of Na⁺-K⁺ ATPase and carbonic anhydrase were measured through the early post-embryonic development of Penaeusjaponicus. In adults, only the Na⁺-K⁺ ATPase activity was measured.
• 2.2. ATPase activity was variable in the successive development stages. From zero in nauplii, the activity slightly increased in zoeae, and rose sharply in mysis stages 2 and 3.
• 3.3. A further significant increase in activity was noted at the transition from late mysis to early postlarvae, concomitant with a change from the larval osmoconforming pattern of osmoregulation to the postlarval and adult hyper-hyporegulating pattern.
• 4.4. The activity of Na⁺-K⁺ ATPase, measured in isolated cephalothorax, increased from PL3 to PL4 to its maximum value in PL5; at this stage, osmoregulatory capacity was fully efficient.
• 5.5. In young stages of P. japonicus, the variations in Na⁺-K⁺ ATPase activity appear correlated with the development of osmoregulatory ultrastructures, and with osmoregulation and salinity tolerance.
• 6.6. These results are discussed with regard to their ecological and physiological implications.
• 7.7. In adults, the activity of Na⁺-K⁺ ATPase was high in gills and epipodites and no activity was detected in branchiostegites. These results are related to the ultrastructure of these organs.
• 8.8. The activity of carbonic anhydrase did not change significantly in larval and postlarval stages.
• 9.9. From these results, it is proposed that the effector sites of osmoregulation are located in branchiostegites, pleurae and epipodites in postlarvae, and in epipodites and mainly in gills in adults.

     

Effects of affinity-purified antibodies on the Ca2+ pumping ATPase of erythrocyte membranes

Antibodies raised in rabbits against the purified erythrocyte membrane Ca²⁺ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg²⁺-ATPase activity. In such cases, the antibodies inhibited the Mg²⁺-ATPase as well as the Ca²⁺-ATPase. This is consistent with the hypothesis that a portion of the Mg²⁺-ATPase activity of ghosts is derived from the Ca²⁺-ATPase. When nitrophenylphosphatase activity was observed, both Mg²⁺ - and Ca²⁺-stimulated activities were observed. Only the Ca²⁺ activity was inhibited by the antibodies, confirming that this activity is due to the Ca²⁺ pump, and suggesting that the Mg²⁺-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca²⁺-ATPases had no effect on the Na⁺-K⁺-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na⁺-K⁺-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na⁺-K⁺-ATPase.In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca²⁺-ATPase, while antibodies added to the outside had no significant effect.     

保幼激素类似物对家蚕丝腺与脂肪体蛋白质合成的调节作用

应用示踪原子法,研究了家蚕Bombyx mori 5龄幼虫丝腺与脂肪体细胞内蛋白质合成的变化规律及保幼激素类似物(JHA738)的调节作用。从5龄初到龄末,家蚕丝腺细胞内蛋白质合成持续升高,5龄中期、后期的蛋白质合成活性分别是前期的1.60倍和2.86倍;全龄出现2个合成高峰,一个是在5龄72h,为细胞固有蛋白质合成高峰,另一个是在5龄192h,为丝蛋白合成高峰。脂肪体细胞内蛋白质合成作用呈现脉冲式的变化。在5龄前期和中期用JHA处理家蚕(剂量为4μg/条),对丝腺细胞固有蛋白质合成和脂肪体细胞蛋白质合成均表现出抑制作用,而对丝蛋白合成则表现出促进作用。本实验结果为进一步阐明JHA增丝机理提供了直接证据。     

Studies on plasma membranes. XXI. Inhibition of liver plasma membrane enzymes by tumour-promoting phorbolester, mitotic inhibitors and cytochalasin B

The tumour promotor tetradecanoyl phorbol acetate (TPA) inhibited the Mg²⁺-, Ca²⁺- and (Na⁺-K⁺)ATPases of rat-liver plasma membranes. A nonpromoting phorbolester derivative was without effect. Colchicine and/or vinblastine inhibited the (Na⁺-K⁺)ATPase, glucagon-stimulated adenylate cyclase, and cyclic adenosine-3, 5-monophosphate (c-AMP) phosphodiesterase, but were without significant effect on the Mg²⁺- or Ca²⁺-ATPase. Cytochalasin B inhibited the (Na⁺-K⁺)ATPase. The results furnish the first direct evidence that these drugs may interact with plasma membranes. The mechanism of the enzyme inhibitions is briefly discussed.     

Localization of a Mg2+-Activated ATPase in the Plasma Membrane of Trypanosoma cruzi1

The Wachstein and Meisel incubation medium was used to detect ATPase activity in epimastigote, spheromastigote (amastigote), and bloodstream trypomastigote forms of Trypanosoma cruzi. Reaction product, indicative of enzyme activity, was associated with the plasma membrane covering the cell body and the flagellum of the parasite. No reaction product was found in the portion of the plasma membrane lining the flagellar pocket. The plasma membrane-associated ATPase activity was not inhibited by ouabain or oligomycin, was detected in incubation medium without K⁺, was inhibited by prolonged glutaraldehyde fixation, and its activity was diminished when Mg²⁺ was omitted from the incubation medium. The Ernst medium was used to detect Na⁺-K⁺-ATPase activity in T. cruzi. No reaction product indicative of the presence of this enzyme was detected. Reaction product indicative of 5'-nucleotidase was not detected in T. cruzi. Acid phosphatase activity was detected in lysosomes. These results indicate that a Mg²⁺-activated ATPase is present in the plasma membrane of T. cruzi and that it can be used as an enzyme marker, provided that the mitochondrial and flagellar ATPases are inhibited, to assess the purity of plasma membrane fractions isolated from this parasite.     

Variations in myocardial CPK and Na+-K+ ATPase following normo- and hypothermic exposure to dimethyl sulfoxide and glycerol.

Exposure of canine myocardial tissue homogenates to Me₂SO glycerol (20 to 60%) for periods up to 8 hr resulted in significant alterations in enzyme activity at 0 °, 18 °, and 37 °C. Both CPK and Na⁺-K⁺ ATPase demonstrate anomalous enhancement of activity at each temperature with glycerol. Me₂SO provides a similar enhancement of Na⁺-K⁺ ATPase activity at hypothermic temperatures up to 40%. Thereafter, nearly complete inhibition resulted. Under normothermic conditions complete Me₂SO inhibition occurred at 40 °. CPK activity diminished in a linear fashion after 4 hr at 18 ° and 37 ° but was unaffected by up to 40% Me₂SO at 0 °C. The results suggest that disruption of the CPK-Na⁺-K⁺ ATPase systems may be minimized by hypothermic perfusion at low cryoprotectant concentrations.     

Comparison of lipid effects on K+-Mg2+ activated p-nitrophenyl phosphatase and Na+-K+-Mg2+ activated adenosine triphosphatase of membrane

Abstract— A comparison was made between K⁺-Mg²⁺ activated p-nitrophenyl phosphatase and Na⁺-K⁺-Mg²⁺ activated adenosine triphosphatase with a solubilized enzyme preparation from a membrane fraction of cerebral cortex. The NPPase showed activity even in the absence of phospholipid, whereas the ATPase required the lipid for its activity. More varied types of phospholipids were effective in activating the NPPase than the ATPase, and with each phospholipid the extent and the pattern of the NPPase activation differed from that of the ATPase. By deoxycholate treatment the pH optimum of the NPPase was shifted independently from the pH optimum shift of the ATPase. The specific activity ratio of the NPPase to the ATPase was not constant during purification. These two enzymes were, however, not separable with ammonium sulphate fractionation, and their thermo-lability was identical regardless of the presence of phospholipid. The results suggested two possibilities: (1) the NPPase is a separate enzyme entity from the ATPase; (2) although the NPPase is a part of the ATPase system, the mechanism of action of lipids on the former part differs from that on the rest of the system.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

镉胁迫对家蚕脂肪体脂质过氧化物含量及抗氧化酶活性和mRNA表达的影响

【目的】探讨鳞翅目模式昆虫家蚕Bombyx mori作为重金属污染的监测指示生物在镉胁迫下的酶反应及相关的基因表达。【方法】给家蚕幼虫期全龄添食镉(Cd²⁺), 调查不同性别家蚕5龄幼虫脂肪体中脂质过氧化物丙二醛（MDA）的含量, 超氧化物歧化酶（SOD）、 过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）的活性及其基因表达水平的变化。【结果】Cd²⁺胁迫对雌雄家蚕MDA 含量均具有浓度效应关系, MDA含量随Cd²⁺胁迫浓度的升高而增加。Cd²⁺胁迫下, SOD和CAT活性表现为先升后降的变化趋势, Pearson相关性分析显示SOD和CAT活性变化有显著相关性(雄: R=0.770, P=0.001; 雌: R=0.854, P=0.000）。雌性家蚕脂肪体中CAT活性变化和Cat mRNA水平的表达具有正相关性（R=0.712, P=0.003）。雄性家蚕脂肪体中GSH-Px活性随Cd²⁺胁迫浓度的升高而增加, 显示浓度 效应关系, 12.5~50 mg/kg Cd²⁺胁迫组GSH-Px活性与对照相比有显著差异（P<0.05）, 其活性和GSH-Px mRNA水平的表达具有正相关性（R=0.834, P=0.000）; 雌性家蚕脂肪体中GSH-Px活性表现为先升后降的变化趋势, 12.5 mg/kg Cd²⁺胁迫组GSH-Px活性与对照相比有显著增加（P<0.01）。【结论】结果表明, 急性镉胁迫对家蚕脂肪体有明显的毒性作用, 其作用机制与脂质过氧化加剧和抗氧化酶活性变化有关。家蚕对重金属镉的解毒机制有性别相关性。     

Variations in myocardial CPK and Na+-K+ ATPase following long-term freezing.

The activity of the soluble enzyme CPK and the membrane bound enzyme Na⁺-K⁺ ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at ?196 °C and ?79 °C and falls off during one month storage at ?40 °, ?20 °, and 0 °C. Na⁺-K⁺ ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.