scholarly journals The hydroxylation of tyrosine by an enzyme from third-instar larvae of the blowfly Calliphora erythrocephala.

1975 ◽  
Vol 147 (3) ◽  
pp. 565-573 ◽  
Author(s):  
R N Pau ◽  
C Kelly
Keyword(s):  
Phenol Oxidase ◽  
Enzyme Concentration ◽  
Polyacrylamide Gels ◽  
Dihydroxybenzoic Acid ◽  
Calliphora Erythrocephala ◽  
Glycine Buffer ◽  
Hydrogen Donors ◽  
Lag Period ◽  
Tyrosine Concentration ◽  
Third Instar Larvae

1. Two pro-(phenol oxidase) were distinguished when the blood of late-third-instar larvae of Calliphora erythrocephala was electrophoresed in polyacrylamide gels with Tris-glycine buffer, pH 8.3. One pro-(phenol oxidase), after activation by an enzyme readily catalyses the oxidation of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-dopa). The second enzyme catalyses the oxidation of L-dopa but not of L-tyrosoine. 2. One of the pro-(phenol oxidases) was purified over 2000-fold from homogenates of whole larvae. This enzyme, after activation, catalyses the oxidation of both dopa and tyrosine. On electrophoresis in polyacrylamide gels with Tris-glycine buffer, pH 8.3, it has the same mobility as the enzyme in the blood which catalyses the oxidation of both tyrosine and dopa. 3. The pro-(phenol oxidase)-activating enzyme was purified over 100-fold from homogenates of whole larvae. 4. The oxidation of L-tyrosine, in the presence of the activated purified phenol oxidase, reached a steady maximum rate after a lag period that was directly related to tyrosine concentration and inversely related to enzyme concentration. 5. The effect of the addition of electron donors on the lag period was studied. Dopa, dopamine (3,4-dihydroxyphenethylamine) and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine are the most effective hydrogen donors. 3,4-Dihydroxybenzoic acid, the oxidation of which was not catalysed by the activated pro-(phenol oxidase), did not affect the lag period.

Use of Platelet Aggregometer to monitor the chymosin-initiated coagulation of casein micelles

1986 ◽  
Vol 53 (3) ◽  
pp. 359-370 ◽  
Author(s):  
Neal A. Bringe ◽  
John E. Kinsella
Keyword(s):  
Light Transmission ◽  
Average Rate ◽  
Enzyme Concentration ◽  
Casein Micelle ◽  
Clotting Time ◽  
Casein Micelles ◽  
Catalysed Reaction ◽  
High Enzyme ◽  
Lag Period ◽  
Limiting Value

SUMMARYThe chymosin-initiated coagulation of casein micelles was followed by monitoring light transmission using a Platelet Aggregometer. The release of macropeptide by chymosin was monitored using fluorescamine. The lag period in the clotting reaction was proportional to clotting time and the reciprocal of enzyme concentration. The average rate of coagulation, which was approximately equal to the reciprocal of clotting time (Tc), increased in proportion to enzyme concentration at low enzyme concentrations and reached a limiting value at high enzyme concentrations. The percentage hydrolysis at the Tc was 47 ± 5% in the presence of 20 mM-CaCl2 and it was calculated that a 5-fold decrease in the speed of the enzyme-catalysed reaction would decrease this value at the Tc to 43 ± 5%. The possible uses and limitations of the Platelet Aggregometer for determining the influence of the chemical environment on the velocity of the chymosin-catalysed reaction and para-casein micelle aggregatability are discussed.

Specific inhibition by ATP and other properties of an endonuclease of Neurospora crassa

1968 ◽  
Vol 46 (10) ◽  
pp. 1285-1291 ◽  
Author(s):  
E. Z. Rabin ◽  
M. Mustard ◽  
M. J. Fraser
Keyword(s):  
Neurospora Crassa ◽  
Nuclease Activity ◽  
Enzyme Concentration ◽  
Specific Inhibition ◽  
Endonuclease Activity ◽  
Dna And Rna ◽  
Period 2 ◽  
Lag Period ◽  
Ageing Period ◽  
Soluble Material

An endonuclease specific for single-stranded DNA and RNA was purified from Neurospora crassa conidia by the method of Linn and Lehman (J. Biol. Chem. 240, 1287 (1965)). The activity of the enzyme was measured by following the rate of release of acid-soluble material at 37 °C from either denatured (single-stranded) or native DNA. After an initial lag period, the length of which was inversely proportional to enzyme concentration, the release of acid-soluble material occurred at a rate which was directly proportional to enzyme concentration. Freshly purified enzyme catalyzed the release of acid-soluble material from denatured DNA 50–70 times faster than from native DNA. This ratio of activities increased to approximately 200 following storage at 0–4 °C. Throughout this "ageing" period 2 × 10−4 M ATP inhibited the activity of the enzyme toward both denatured and native DNA by 50%. The following nucleoside phosphates, at a concentration of 4 × 10−4 M, had no effect on the activity of the endonuclease toward denatured DNA: ADP, AMP, ITP, XTP, CTP, dTTP, and UTP. GTP at 4 × 10−4 M inhibited this activity by 15%. Both ATP and dATP at 1 × 10−4 M inhibited endonuclease activity against denatured DNA by about 25%. The inhibition by ATP was noncompetitive over the range of concentration 0.11–0.75 mg DNA per milliliter. Specific inhibition of nuclease activity by ATP has not been previously reported.Several other properties of the endonuclease were examined. There is evidence that endonuclease action was inhibited by accumulation of the products of digestion. The enzyme lost activity toward denatured DNA in a medium containing 0.3 M NaCl but activity was partially restored in the presence of 4 × 10−3 M mercaptoethanol. The endonuclease did not digest DNA–RNA hybrid to any appreciable extent.

Notes upon the biology and morphology of the immature stages of Neottiophilum praeustum (Meigen, 1926)(Diptera: Neottiophilidae) parasitic on birds

Parasitology ◽  
1954 ◽  
Vol 44 (1-2) ◽  
pp. 111-119 ◽  
Author(s):  
P. Tate
Keyword(s):  
Feeding Habits ◽  
Instar Larva ◽  
Rhagoletis Pomonella ◽  
Immature Stages ◽  
Primitive Character ◽  
Prepupal Stage ◽  
Calliphora Erythrocephala ◽  
The Third ◽  
Third Instar Larvae

1. The feeding habits of second- and third-instar larvae of Neottiophilum praeustum have been observed and show that this species is a true parasite of birds and feeds by sucking the blood of nestlings.2. If they are too numerous the larvae may kill the nestlings. Although they will continue to feed upon dead birds, and even penetrate into the viscera, such food is unsuitable for the development of the larvae and they become greatly distended and die within a few days.3. The morphology of the hitherto unknown second-instar larva is described and is compared with that of the third instar.4. Within the puparium of Neottiophilum praeustum there is a fourth moult resulting in the formation of a cast prepupal cuticle which resembles that described by Snodgrass in Rhagoletis pomonella and is much better developed than the prepupal cuticle in Calliphora erythrocephala.5. The better development of the prepupal cuticle in the acalypterates than in calypterates indicates that the presence of a prepupal stage in the cyclorrhaphous Diptera is a primitive character and is progressively reduced until in the higher families it is almost vestigial.

The action of calf rennet and other proteolytic enzymes on κ-casein

1969 ◽  
Vol 36 (1) ◽  
pp. 11-20 ◽  
Author(s):  
R. C. Lawrence ◽  
L. K. Creamer
Keyword(s):  
Protein Interactions ◽  
Proteolytic Enzymes ◽  
Enzyme Concentration ◽  
Ion Concentration ◽  
Protein Protein Interactions ◽  
Low Concentrations ◽  
Lag Period ◽  
High Concentrations ◽  
The Stability ◽  
Calcium Ion Concentration

SummaryThe hydrolysis of κ-casein by a number of rennets and other proteolytic enzymes has been followed by measuring the increase in opacity due to the formation of insoluble aggregates of para-κ-caseins. The stability of these precipitates varied markedly, some being solubilized rapidly by the further action of the enzyme. The turbidity obtained with certain enzymes was dependent upon the calcium ion concentration, indicating that the para-κ-caseins produced were not identical for all enzymes.For high concentrations of calf rennet, the rate of aggregation was linear with respect to time. With low concentrations of enzyme, increase in turbidity was preceded by a lag period which was lengthened by decreasing the enzyme concentration or increasing the κ-casein concentration. This increase in lag is favoured by a high κ-casein/para-κ-casein ratio, suggesting that the aggregation of newly formed para-κ-casein is prevented by the unchanged κ-casein. In addition, small amounts of αs1- or β-caseins present in the κ-casein also markedly affected the aggregation of para-κ-casein, indicating that all 3 major casein components can inhibit the aggregation of para-κ-casein in the absence of calcium ions. In the light of these observations the possible role of protein-protein interactions in casein coagulation by calf rennet is discussed.

scholarly journals The isolation of an o-diphenal oxidase from third-instar larvae of the blowfly Calliphora erythrocephala

1975 ◽  
Vol 149 (3) ◽  
pp. 707-712 ◽  
Author(s):  
R N Pau ◽  
P A M. Eagles
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Sucrose Density Gradient ◽  
Crystalline Fraction ◽  
Calliphora Erythrocephala ◽  
A Minor ◽  
Third Instar Larvae

1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.

scholarly journals A generalized theory of the transition time for sequential enzyme reactions

1981 ◽  
Vol 199 (1) ◽  
pp. 155-161 ◽  
Author(s):  
J S Easterby
Keyword(s):  
Steady State ◽  
Enzyme Concentration ◽  
Transition Time ◽  
Successful Operation ◽  
Enzyme Reactions ◽  
Minimum Requirements ◽  
Lag Period ◽  
State Production ◽  
Definition Of ◽  
Simple Systems

In a sequence of coupled enzyme reactions the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. Provided that a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaelis-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Application of the theory to coupled enzyme assays allows a definition of the minimum requirements for successful operation of the assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration.

Fine Structure of the Malpighian Tubules of the Mosquito, Culex pipiens

1971 ◽  
Vol 29 ◽  
pp. 402-403
Author(s):  
Brendan Clifford
Keyword(s):  
Fine Structure ◽  
Culex Pipiens ◽  
Stellate Cell ◽  
Malpighian Tubule ◽  
Cell Types ◽  
Instar Larva ◽  
Malpighian Tubules ◽  
Calliphora Erythrocephala ◽  
Distinct Cell ◽  
Basal Plasma

An ultrastructural investigation of the Malpighian tubules of the fourth instar larva of Culex pipiens was undertaken as part of a continuing study of the fine structure of transport epithelia.Each of the five Malpighian tubules was found to be morphologically identical and regionally undifferentiated. Two distinct cell types, the primary and stellate, were found intermingled along the length of each tubule. The ultrastructure of the stellate cell was previously described in the Malpighian tubule of the blowfly, Calliphora erythrocephala by Berridge and Oschman.The basal plasma membrane of the primary cell is extremely irregular, giving rise to a complex interconnecting network of basal channels. The compartments of cytoplasm entrapped within this system of basal infoldings contain mitochondria, free ribosomes, and small amounts of rough endoplasmic reticulum. The mitochondria are distinctive in that the cristae run parallel to the long axis of the organelle.

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