Enzymic oxidation of some alkylbenzenes in insects and vertebrates

1. Oxidation rates of alkylbenzenes have been measured in 10000g supernatants of vertebrate livers, locust fat bodies and housefly abdomens. 2. Activity per g. of insect was greater in fly than locust preparations but both were of the same order as a range of vertebrate species. 3. Methyl groups of toluene and p-nitrotoluene were oxidized more rapidly than the side chains of higher homologues. 4. In the higher homologues hydroxylation occurred most readily at the α-methylene group and less readily at penultimate methylene and terminal methyl groups. 5. Oxidations in both vertebrates and insects were inhibited by piperonylbutoxide and similar synergists. 6. Oxidation activity was stimulated by pretreatment of rats, but not locusts, with phenobarbitone or 3,4-benzopyrene.     

Detection and determination of Locusta migratoria trypsin by radioimmunoassay

Polyclonal antibodies were produced against trypsin-like enzyme TLE-2, one of two trypsins isolated from the midgut cecae of the locust Locusta migratoria. Using those antibodies a heterologous RIA was developed. The specificity of the antibodies was demonstrated by immunoblot technique and cross-reactivity experiments. Positive blot reaction with the antiserum was observed with locust trypsins and binding competition was obtained only with the second trypsin from the locust, TLE-1. Trypsins from bovine and hog as well as trypsins from Tenebrio molitor and Tribolium castaneum and locust chymotrypsin did not inhibit TLE-2 binding to the antiserum even at concentrations 1,000-fold greater than that of locust TLE-2. The concentrations of TLE-2 in cecal fluid, cecal wall, hemolymph, and fat-body were 2,790, 60.4, 6.4, and 0.7 ng per mg tissue, respectively. © 1992 Wiley-Liss, Inc.     

A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

A comparison of the properties of the phosphofructokinases of the fat body and flight muscle of the adult male desert locust

1. Phosphofructokinase was isolated, and partially purified by ammonium sulphate fractionation, from the fat body and flight muscle of the desert locust. 2. Ammonium sulphate appears to stabilize the enzymes, but does not activate them. 3. Both flight-muscle and fat-body enzymes give sigmoidal hexose monophosphate concentration-activity curves, which are characteristic of regulatory enzymes. 4. At low ATP concentrations both the enzyme activities increase rapidly with increasing ATP concentrations, but above an optimum concentration ATP becomes inhibitory. This optimum concentration is 0.2mm for the fat-body enzyme and 0.1mm for the flight-muscle enzyme. 5. AMP activates both the enzymes; half-maximal activation occurs at 10mum in each case, the effect being independent of substrate concentration. 6. 3',5'-(cyclic)-AMP (0.5mm) and P(i) (1mm) activate the flight-muscle enzyme, but have no effect on the fat-body enzyme. 7. FDP (1mm) inhibits both enzymes, and with the flight-muscle enzyme this inhibition is increased by increasing the ATP concentration. 8. Citrate, phosphoenolpyruvate and alpha-glycerophosphate have no effect on either enzyme under the assay conditions used. 9. The properties of phosphofructokinases from the locust are compared with those of phosphofructokinases from other sources.     

Vitellogenin in locusts (Locusta migratoria): Translation of vitellogenin mRNA in Xenopus oocytes and analysis of the polypeptide products

Cytoplasmic poly(A)⁺-RNA, prepared from fat bodies of reproductively active locust females, directed the synthesis of two large polypeptides in Xenopus oocytes. Occasionally two smaller polypeptides (X₁ and X₂) were also detectable. The two larger polypeptides were immunologically and electrophoretically indistinguishable from the unprocessed Vitellogenin polypeptides (Vg₁ and Vg₂) of locust fat bodies. Peptide patterns generated from these two translation products by Staphylococcus aureus V8 protease digestion were identical to those of Vg₁ and Vg₂. RNA isolated from allatectomized female locusts treated with the juvenile hormone analog (ZR-515) was also able to direct the synthesis of vitellogenin in Xenopus oocytes, whereas RNA isolated from mature males or allatectomized females did not. The molecular weights of fat-body Vg₁ and Vg₂ were 235,000 and 225,000, respectively and the processed vitellogenin polypeptides were found to range from 126,000 to 52,000. Electrophoretic and Chromatographic analysis of [³⁵S]methionine-containing tryptic peptides of Vg₁ and Vg₂ showed two different tryptic peptide fingerprints. Distinctly different peptide patterns were also observed when Vg₁ and Vg₂ were partially digested with V8 protease or papain. However, tryptic peptide mapping and V8 protease limited digestion mapping of fat-body X₁ and X₂ revealed that these two polypeptides were derived from Vg₁ and Vg₂. This suggests that Vg₁ and Vg₂ are products of two different vitellogenin structural genes.     

A Spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH₂ and oxygen. Activity with NADH₂ was one-third of that with NADPH₂. 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at −12°. Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2′-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.     

Purification and some properties of steryl β-d-glucoside hydrolase from Sinapis alba seedlings

Homogenates of 7-day-old S. alba seedlings hydrolysed cholesteryl[4-H¹⁴C] β-d-glucoside or sitosteryl β-d-glucoside-[6-³H]. Activity was located predominantly in the cell membrane structures sedimenting at 1000–15 000 g and was solubilized by acetone treatment. Partially purified enzyme preparation, with an about 1500 times higher specific activity with respect to the crude homogenate, was obtained by repeated acetone precipitation and subsequent chromatography on DEAE-Sephadex and Sephadex G-100. During this procedure a considerable separation from other enzymes with β-glucosidase activity was achieved. The enzyme had MW 65 000 daltons, pH optimum at 5.2–5.6. Two observations suggested that the enzyme was a specific steryl β-d-glucoside hydrolase. Firstly, there was no substrate competition between steryl glucosides and several other β-d-glucosides. Secondly, enzyme activity wasstrongly inhibited by low concentrations of various 3β-OH sterols with a planar ring system and an intact side chain.     

Geranylpyrophosphate: p-hydroxybenzoate geranyltransferase activity in extracts of Lithospermum erythrorhizon cell cultures

The enzymatic formation of m-geranyl-p-hydroxybenzoic acid from geranylpyrophosphate and p-hydroxybenzoic acid was investigated in cell-free extracts of Lithospermum erythrorhizon cell cultures. The reaction required the presence of a divalent cation, magnesium being the most effective activator. The enzyme showed a very broad pH optimum between pH 7.1 and 9.3. It was highly specific for both p-hydroxybenzoic acid and geranylpyrophosphate, and the apparent K_m values for these two substrates were 0.014 and 0.56 mM, respectively. The activity was located in the pellet of a 100 000 g centrifugation, showing that the enzyme is bound to membranes or microsomes. Shikonin-producing cultures contained an activity of this enzyme 35 times higher than non-producing cultures, suggesting that this enzyme is of regulatory importance in shikonin biosynthesis.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

Synthesis of 4-carboxy-2-polyprenylphenols by a particulate fraction of baker's yeast

The preparation ofa cell-free homogenate and 10000 g particulate fraction with polyprenylpyrophosphate-p-hydroxybenzoate polyprenyltransferase activity from 0 to 7-day-old blocks of compressed baker's yeast is described. The synthesis of 4-carboxy-2-triprenylphenol from p-hydroxybenzoate and FPP by the particulate fraction has been studied in some detail. In particular it has been shown that the transferase catalysing the reaction is activated by Mg²⁺, has a pH optima of 7 and is inhibited by phosphate buffer. Intracellular distribution studies have established that in freshly grown cells of Saccharomyces carlsbergensis the greater part of the polyprenyl transferase activity is present in the mitochondria.     

Specificity of aspartate aminotransferases from leguminous plants for 4-substituted glutamic acids

Aspartate aminotransferase (glutamate-oxalacetate transaminase) was partially purified from extracts of germinating seeds of peanut (Arachis hypogaea), honey locust (Gleditsia triacanthos), soybean (Glycine max), and Sophora japonica. The ability of these enzyme preparations, as well as aspartate aminotransferase purified from pig heart cytosol, to use 4-substituted glutamic acids as amino group donors and their corresponding 2-oxo acids as amino group acceptors in the aminotransferase reaction was measured. All 4-substituted glutamic acid analogs tested were poorer substrates than was glutamate or 2-oxoglutarate. 2-Oxo-4-methyleneglutarate was least effective (lowest relative V_m/K_m) as a substrate for the enzyme from peanuts and honey locust, which are the two species studied that accumulate 4-methyleneglutamic acid and 4-methyleneglutamine. Of the different aminotransferases tested, the enzyme from honey locust was the least active with 2-oxo-4-hydroxy-4-methylglutarate, the corresponding amino acid of which also accumulates in that species. These results suggest that transamination of 2-oxo-4-substituted glutaric acids is not involved in the biosynthesis of the corresponding 4-substituted glutamic acids in these species. Rather, accumulation of certain 4-substituted glutamic acids in these instances may be, in part, the result of the inefficacy of their transamination by aspartate aminotransferase.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

Characteristics and intracellular distribution of messengerlike RNA in encysted embryos of Artemia salina

Homogenates of dormant cysts of Artemia salina were fractionated by differential centrifugation. RNA was prepared from the various fractions and tested for stimulatory activity in a [¹⁴C]leucine incorporating Escherichia coli system. The highest specific activity was found in the RNA extracted from a cytoplasmic fraction sedimenting at 15,000 g. Some activity was associated with the soluble and crude ribosomal fractions, while the RNA extracted from the crude nuclear fraction was less active.The 15,000 g sediment was purified by centrifugation in a sucrose density gradient. The active material formed a characteristic, colored band at a buoyant density of about 1.17 g/ml. The banding fraction was mainly composed of endoplasmic vesicles and mitochondria. The specific activity of the extracted RNA was further increased when the 15,000 g sediment was treated with buffered 20–100 mM EDTA (with or without 0.1% Triton X-100) before banding.Sedimentation analysis of the active RNA from the purified 15,000 g fractions revealed three distinct absorption peaks at 28 S, 18 S, and 16 S, apparently representing cytoplasmic and mitochondrial rRNA. The 28 S and 18 S peaks were reduced by EDTA treatment, but only to a certain limit. By gel electrophoresis a number of additional components were resolved, including 4 S and 5 S RNA. The template activity showed a heterodisperse distribution with a maximum at 17–20 S, not correlated with the 16 S peak. Isolated 18 S and 28 S rRNA had very low activity.The experiments suggest that in Artemia cysts an appreciable amount of messengerlike RNA is associated with mitochondria and/or endoplasmic vesicles carrying ribosomal monomers.     

α-Galactosidase from sweet chestnut seeds

The major sugars of fresh seeds of Castanea sativa were shown to be raffinose, stachyose and sucrose. Drying seeds at 25° for 14 weeks increased the ratio raffinose: stachyose from 1.1 to 3.5, reduced sucrose content by ca 50 % and decreased total extractable α-galactosidase. The enzyme activity was resolved into two peaks, a high MW form I (apparent MW215 000) and a low MW form II (apparent MW 53 000). The latter form was predominant in the extract of fresh seeds whereas the former was the main form in the 14-week dried seeds. An increase in the amount of enzyme I was also observed when a buffered extract (pH 5.5) of fresh seeds was stored at 4°. Enzymes I and II had pH optima of 4.5 and 6, respectively. Both enzymes hydrolysed p-nitrophenyl α-d-galactoside at a much greater rate than the natural substrates raffinose, stachyose, locust bean gum and carob gum. However, enzyme I showed preference for stachyose as compared to raffinose; the opposite order was observed for enzyme II.     

Cinnamic acid 4-hydroxylase from gherkin tissues

Homogenates from 4-day-old gherkin cotyledons and hypocotyls fractionated by sucrose density gradient centrifugation contain cinnamic acid 4-hydroxylase activity, the activity being highest in the endoplasmic reticulum fractions. These fractions also contain very low concentrations of cytochrome P₄₅₀. Hydroxylase activity is dependent on NADPH and on molecular oxygen, is optimal at pH 7.5 and is inhibited by carbon monoxide. The enzyme is very sensitive to inhibition by 2-mercaptoethanol, but it is not inhibited by the product, p-coumaric acid. Further, its responses to various potential inhibitors are fairly typical of mixed function oxidases from other sources.     

Intracellular distribution of NADP-linked isocitrate dehydrogenase,fumarase and citrate synthase in bovine heart muscle

By fractional extraction of minced bovine heart muscle with iso-osmotic sucrose and phosphate buffer solutions, it is shown that less than 4% of the total citrate synthase in the tissue is in the cytosol. Using citrate synthase as a marker for broken mitochondria, two methods of fractionation of 750 × g supernatants from homogenates of bovine heart muscle show that 10% of the total fumarase and NADP-linked isocitrate dehydrogenase activities are present in the cytoplasm. Homogenates prepared by sonication and osmotic shock and by sand-grinding gave closely similar results as regards enzyme distributions and extent of mitochondrial breakage. The results are compared with those reported for other tissues.     

Cloning and characterization of a modular GH5 β-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13

The gene (1272-bp) encoding a β-1,4-mannanase from a gut bacterium of Eisenia fetida, Cellulosimicrobium sp. strain HY-13 was cloned and expressed in Escherichia coli. The recombinant β-1,4-mannanase (rManH) was approximately 44.0 kDa and has a catalytic GH5 domain that is 65% identical to that of the Micromonospora sp. β-1,4-mannosidase. The enzyme exhibited the highest catalytic activity toward mannans at 50 °C and pH 6.0. rManH displayed a high specific activity of 14,711 and 8498 IU mg⁻¹ towards ivory nut mannan and locust bean gum, respectively; however it could not degrade the structurally unrelated polysaccharides, mannobiose, or p-nitrophenyl sugar derivatives. rManH was strongly bound to ivory nut mannan, Avicel, chitosan, and chitin but did not attach to curdlan, insoluble oat spelt xylan, lignin, or poly(3-hydroxybutyrate). The superior biocatalytic properties of rManH suggest that the enzyme can be exploited as an effective additive in the animal feed industry.     

Regulation of locust fat-body phosphorylase

1. Glycogen phosphorylase of locust fat-body was partially purified by differential centrifugation and dissociation from glycogen particles at two pH values. 2. Optimum activity was obtained at pH6.6-6.7. 3. The calculated apparent K(m) values for glycogen and glucose 1-phosphate were 0.08% and 10-13mm respectively. 4. 5'-AMP activated in the range 5mum-1mm. 5. Glucose 6-phosphate is a competitive inhibitor for the substrate glucose 1-phosphate (K(i)=1.7mm). 5'-AMP abolishes this inhibition. Glucose weakly inhibits (K(i)=25-30mm), but trehalose does not inhibit even at 100mm. 6. It is suggested that glucose 6-phosphate is a major regulator of glycogen phosphorylase activity in locust fat-body.