Cholecystokinin-induced residual stimulation of enzyme secretion from mouse pancreatic acini

When dispersed acini from mouse pancreas are first incubated with cholecystokinin octapeptide, washed and then reincubated with no additions there is significant stimulation of amylase secretion during the second incubation (residual stimulation of enzyme secretion). Cholecystokinin-induced residual stimulation of enzyme secretion is modified, but not abolished, by reducing the temperature of the first incubation from 37 degrees C to 4 degrees C. Measurement of binding of 125I-labeled cholecystokinin octapeptide indicated that maximal cholecystokinin induced residual stimulation of enzyme secretion occurs when 12-20% of cholecystokinin receptors are occupied by cholecystokinin octapeptide. Moreover, maximal cholecystokinin-induced residual stimulation of amylase secretion is 25% greater than maximal cholecystokinin-induced direct stimulation of amylase secretion. Cholecystokinin tetrapeptide, which causes the same maximal direct stimulation of amylase secretion as does cholecystokinin octapeptide, causes a maximal residual stimulation of enzyme secretion that is only 30% of that caused by a maximally effective concentration of cholecystokinin octapeptide. Adding dibutyryl cyclic GMP to the second incubation can reverse the residual stimulation caused by adding cholecystokinin to the first incubation. The pattern and extent of the dibutyryl cyclic GMP-induced reversal of residual stimulation varies, depending on the temperature and concentration of cholecystokinin octapeptide in the first incubation. The present results are compatible with the hypothesis that mouse pancreatic acini possess two classes of cholecystokinin receptors. One class has a relatively high affinity for cholecystokinin and produces stimulation of enzyme secretion; the other class has a relatively low affinity for cholecystokinin and produces inhibition of enzyme secretion.     

Changes in gill histology of fathead minnows and yellow perch transferred to soft water or acidified soft water with particular reference to chloride cells

Summary Fathead minnows, Pimephales promelas, and yellow perch, Perca flavescens, were transferred from moderately soft Lake Superior water (hardness 45mg/l as CaCO₃) to very soft diluted Lake Superior water (hardness 4.5mg/l). Sulfuric acid was added in some treatments by means of a multichannel diluter. In very soft water, chloride cells proliferated in the gills, especially in the epithelium of the secondary lamellae. When exposed to acid, chloride cells were damaged and less abundant in the secondary lamellae, and blood osmolality was reduced at pH 5.0 (x = 188 mOsm/kg, 9 days exposure; normal 280 mOsm/kg) for the minnows and pH 4.1 (x = 218 mOsm/kg, 58 days exposure; normal 329 mOsm/kg) for the perch. Certain chloride cells which form gland-like clusters in the primary lamellae of perch gills showed little damage even at pH 4.1. The present study supports the view that chloride cells proliferate in very soft fresh water to help maintain ionic balances, and that damage to these cells in acidified soft water may be related to diminished ionoregulatory capacity. The greater acid tolerance of chloride cells of, and the higher blood osmolality maintained by, perch could help to explain the greater tolerance of this species to low pH. In some cases, a species' ability to acclimate to very soft water and acidified soft water may depend upon the number, distribution, and physiology of its chloride cells.     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

Extracellular and cell-bound lipase activity in relation to growth of Geotrichum candidum

Summary The imperfect fungus Geotrichum candidum produced extracellular lipase in a basic peptone-salt medium. By adding olive oil or Tween 80 to the basic medium the lipase yields could be enhanced and the maximal yields were found with Tween 80, which resulted in a sixfold increase in extracellular lipase activity as compared with basic medium. During the early phase of growth in medium with olive oil the proportion of cell-bound activity was higher than that of extracellular activity, and a delay in the secretion of extracellular lipase was found. The proportion of cell-bound activity from growth in basic medium and in basic medium with Tween 80 was lower than that of extracellular activity during the entire growth phase. Analyses by polyacrylamide gel electrophoresis showed that the lipase activity from growth in all three media could be ascribed to equivalent protein bands at 57 000 and 61 000 daltons. Immunodiffusion showed that the cell-bound preparation contained lipase that was immunologically identical with purified extracellular lipase from G. candidum.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

Algesia and local responses induced by neurokinin A and substance P in human skin and temporal muscle

Neurokinin A (NKA), substance P (SP) and the two peptides combined (SP + NKA) were injected intracutaneously on the forearm and into the temporal muscle of healthy volunteers. Pain intensity, cutaneous wheal and flare responses and tenderness of the temporal muscle were quantitated. SP but not NKA induced cutaneous pain. This relates the algesic effect of SP to the specific N-terminal amino acid sequence of the peptide, not shared by NKA. NKA, however, potentiated the algesic effect of SP as SP + NKA induced a significantly prolonged cutaneous pain sensation. Both peptides induced wheals, but only SP induced flare. These results confirm previous studies relating wheal formation to the identical C-terminal amino acid sequence of the two peptides and flare reaction to the N-terminal part of SP. Injections into the temporal muscle did not cause pain or tenderness.     

Isopenicillin N synthase and desacetoxycephalosporin C synthase activities during defined medium fermentations of Streptomyces clavuligerus: effect of oxygen and iron supplements

When the level of dissolved oxygen was increased to saturation in defined media fermentations of Streptomyces clavuligerus, the total duration of activity of the penicillin ring cyclization enzyme, isopenicillin N synthase (IPNS), was extended by at least 20 h; however, no increase in the stability of the ring expansion enzyme, desacetoxycephalosporin C synthase (DAOCS), was observed. Consequently, the conversion of the excreted intermediate penicillin N to cephamycin C was 15-20% less efficient at this high oxygen concentration. The increased dissolved oxygen level also led to the complete loss of IPNS and DAOCS activities for 4 h during the period of fastest growth, and the rate of specific cephamycin C production fell to zero. A several hundred fold increase in the level of iron in the defined media resulted in a sixfold improvement in the rate of specific cephamycin C production after 60 h fermentation. This increased rate appeared to be due to an elevation in the in vivo activities of a number of the cephamycin biosynthetic enzymes, particularly those catalysing later pathway steps.     

Comparison of transfer RNA and ribosomal RNA intron splicing in the extreme thermophile and archaebacterium Desulfurococcus mobilis

The structure of the exon-intron boundary was compared for an intron within 23S ribosomal RNA of Desulfurococcus mobilis and a newly discovered intron in tRNA(Met) from the same organism. The occurrence of a putative common structural feature suggests that intron excision occurs by the same mechanism. The possible recognition of this structural feature by the cleavage enzyme was investigated for the ribosomal RNA intron using RNA substrates exhibiting various exon and intron deletions. The results support the involvement of the structural features in the cleavage process. The evolutionary implications of these results are considered.     

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Sch?gger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.