Maria L. Bade

Neutral metal chelator-sensitive protease in insect moulting fluid

Abstract Proteolytic activity in moulting fluid from the sphingid Manduca sexta has at least two pH optima; these occur at pH 7 and at pH 7·7. The latter activity is shown to be trypsin-like in that it is susceptible to inhibition by diisopropylfluorophosphate. By contrast, the peak at neutral pH consists of proteolytic activity not hitherto described in invertebrates. This activity shows little or no inhibition with diisopropylfluorophosphate or p -hydroxymercuribenzoate but is strongly inhibited by chelators such as 1,10-phenanthroline, 8-hydroxyquinoline, and EDTA. The neutral metal chelator-sensitive activity requires calcium but the inhibitor data permit the conclusion that the metal ion inhibited by the chelators belongs to the first transition series and thus cannot be calcium. The neutral protease appears to be similar to proteases previously characterized from bacteria and snake venom. In moulting fluid from Manduca , proteolytic activity in vitro is very low in the presence of 1,10-phenanthroline at every pH studied except pH 7·7; in vivo , ecdysis is inhibited in Manduca larvae fed on diet containing a sufficient level (0·02 per cent or higher) of 1,10-phenanthroline. The metal chelator-sensitive proteolytic activity appears to be an essential moulting protease in Manduca .

Time pattern of appearance and disappearance of active molting chitinase in Manduca cuticle. The endogenous activity

Localization of insect molting chitinase in the old Truman’s terminology. An additional check to assure cuticle of pharate Munduca pupae has recently been that the pharate pupae analyzed were developing at reported from this laboratory [l] . It was shown that comparable rates was afforded by the fact that events highly active chitinase is held tenaciously in the cuticle late in the pupal molt, i.e. within the final 32 hr so that its activity remains by comparison low in preceding ecdysis, can be timed approximately by molting fluid and epidermal extracts. Cuticle chitinase observation of the extent of tanning in the new cuticle preparations may release over 200 pg N-acetylglucosamine which at that stage is visible through the old cuticle per milligram cuticle protein per hour at the pH 8.2, of enzymatically active Munduca molting fluid, and were shown to utilize endogenous cuticle chitin preferentially in presence of exogenous colloidal chitin prepared from Manduca cuticles. At the same time, however, endogenous chitinase activity in newly synthesized cuticle was found to be absent. It was of interest, therefore, to determine at what time endogenous chitinase activity arises in insect cuticle relative to the molt. Specific endogenous cuticle chitinase activity rises sharply about 30 hr before pupal ecdysis; it disappears as abruptly about 12 hr later. A similar but lower endogenous peak in old cuticle chitinase activity occurs just prior to the preceding larval molt. Larvae of Munducu sex&, the tobacco homworm, were reared on an artificial diet from eggs obtained through the courtesy of Dr R. A. Bell of the U.S.D.A. Metabolism and Radiation Research Laboratory, Fargo, North Dakota. -The larvae were observed carefully as the 4th larval molt approached, timed with 10 min of ecdysis, and harvested at various times following ecdysis. As previously reported [2] , larval ecdysis of Lepidopteru is associated with a circadian clock. However, animals from the same hatch do not all develop at the same rate, as already described by Truman [2]. The larvae used in the experiments here reported were chosen from the most rapidly developing group, i.e. from those belonging to Gate 1 in

Localization of molting chitinase in insect cuticle

Abstract Chitinase activity in molting larvae of Manduca sexta is localized in old cuticle; it is not quantitatively extracted during homogenization, has good activity at the pH of molting fluid, and preferentially utilizes endogenous cuticle chitin as substrate. It is concluded that cuticle chitinase is the physiologically active molting enzyme in Manduca .

Digestion of cuticle chitin during the moult of Manduca sexta (Lepidoptera: Sphingidae)

At the time of moulting, the old cuticle of Manduca sexta acquires the ability to break down its constituent chitin. The appearance of such endogenous moulting chitinase activity in vivo is not related to the occurrence of apolysis but results from exposure to moulting fluid; chitinase activity is induced in intact or deproteinized cuticle in vitro by incubation with enzymatically active moulting fluid (Bade, 1970). Acquisition of endogenous chitinase activity by the chitin of intact cuticle in vitro requires several steps, one of which involves an enzymatic reaction which is a function of both temperature and concentration of moulting fluid. The process of rendering cuticle chitin able to serve as substrate for chitinase, can be inhibited by anti-trypsin agents and is postulated to be a novel variant of the biological activation of a biopolymer by a protease. Moulting fluid chitinase becomes attached to deproteinized chitin in vitro. Attachment occurs very rapidly, and the extent of this attachment with time is a function of temperature. Digestion of old cuticle during the moult is shown to be an orderly process which comprises a number of steps in sequence; some steps probably overlap in part.

Molting fluid chitinase: A homotropic allosteric enzyme

Abstract The molting fluid of the tobacco hornworm has chitinase activity which shows allosteric behavior with chitin. A parabolic curve is obtained on a double reciprocal plot of 1 v vs. 1 [S] , and a sigmoid curve results when v (N-acetylglucosamine produced) is plotted against [S] (chitin concentration in terms of N-acetylglucosamine concentration). The Hill coefficient with insect chitin is 1.95 ± 0.08. Allostery of the chitinase enables the insect to exert additional control over the integrity of structure of its cuticle.

Activation of old cuticle chitin as a substrate for chitinase in the molt of Manduca.

Abstract During the molt, chitin in the old cuticle of Manduca is digested by chitinase taken up from molting fluid, but the chitin in intact (= premolt) cuticle is not accessible to chitinase. As a prerequisite of digestion, old cuticle chitin is rendered competent to serve as chitinase substrate in a reaction attributable to trypsin-like proteolytic activity of molting fluid.

Biochemistry of insect differentiation: A system for studying the mechanism of chitinase activity in vitro

Abstract Results obtained with an in vitro system for the study of chitinase are described. The system involves soluble enzyme protein(s) and an insoluble substrate preparation. With insect molting fluid chitinase, it shows properties that parallel those observed during in vivo breakdown of cuticle during the molt. For example, molting fluid chitinase activity not previously exposed to chitin is stronly and specifically adsorbed to the substrate, in contrast to other enzymatic activities including hexosaminidase (chitobiase) present in molting fluid. This leads to partial purification of molting fluid chitinase activity reflected in increased specific activity of chitinase associated with the insoluble chitin substrate; we have previously reported increase of specific chitinase activity of (deproteinized) cuticle resulting from its incubation with molting fluid ( M. L. Bade and A. Stinson, 1978 , Biochem. Biophys. Res. Commun. 84 , 381–388). Soluble end product is generated rapidly and linearly with time by the in vitro system; the end product is assumed to be N -acetylglucosamine since the specific radioactivity of this compound is unchanged during the 10 min required for assay. Molting fluid chitinase activity may involve a number of polypeptides ranging in molecular weight from 145,000 to less than 20,000 daltons. The system described gives results consistent with a processive mechanism for molting fluid chitinase, i.e., data are given demonstrating that molting fluid chitinase continues to act on the same chitin particle(s) with which it initially associates rather than diffusing freely from substrate particle to substrate particle, and the product of its action appears to be a monosaccharide rather than a mixture of oligosaccharides. Processive behavior for chitinase would be predicted from the known structure, and the in vivo measured rate of breakdown, of cuticle chitin during the molt; the preliminary nature of this conclusion, based on what is so far known about the structure of the substrate used in the in vitro system, is briefly discussed.

Biochemistry of insect differentiation. Requirements for high in vitro moulting fluid chitinase activity

Abstract A method for preparing chitinase substrate is described; the substrate reproducibly gives high chitinase activity under suitable conditions. For moulting fluid chitinase, these are: a chitin concentration of at least 2 mg/ml, pH 6.5, and presence of calcium; addition of an inhibitor for neutral protease is helpful. Activity may be measured in minutes, either colourimetrically or by counting acid-soluble product from in vivo labelled chitin. N- Acetylglucosamine is the chief soluble end product. Moulting fluid chitinase appears to form a complex with chitin and calcium which successfully competes with EGTA for calcium; the dissociation constant is therefore at least as low as 10 −7 M. A transient increase in product release of chitinase accompanies the first interaction of chitin, chitinase and calcium ions.

Protein assay in the microgram range: Modifications of the hiraoka-glick method☆

Abstract A method is described for the accurate estimation of protein samples ranging in size from 1 to 100 μg. It is based on fluorescence quenching of eosin Y by protein and modifies a method originally published by Hiraoka and Glick. The modified method is convenient to use. It is carried out with a stabilized reagent in cuvettes of standard size and permits the estimation of 5 μg or less of protein with precision of ± 9%. The measurements are shown to be sensitive to slight variations in ambient temperature and these contribute at least 3% to the error term shown. A graphical manner of presenting the data has been devised that provides a linear relationship over nearly 50-fold range of protein concentrations. Quenching is unaffected by presence of glucose or urea and partially inhibited by glycine or mercaptoethanol only if these are present in better than 100-fold excess over protein. Different proteins show different degrees of quenching and the method is thus most suitable for comparing quantitatively protein mixtures of similar composition. The modified method should be useful not only where microgram quantities of protein must be measured, but also with automated equipment or in cases where the Folin-Ciocalteau method cannot be employed due to presence of interfering substances.

Chitin Structure and Chitinase Activity: Isolation of Structurally Intact Chitins

Assessment of chitinase kinetics and mechanism in vitro has been hampered by lack of suitable substrates. We have previously reported rapid linear initial chitinase velocity with chitin substrate isolated from insect larval cuticle. Such chitin is shown to be fibrous in the light microscope. Methods are described for preparing fibrous chitins from any animal source including calcified carapaces. Evidence is given that chitin native fine structure in situ is maintained by structural proteins which in the fibrous chitin isolates are functionally replaced by covalently bound ester groups. Chitin fiber analogues thus reconstructed appear to have retained their native fine structure.