Hypotrypsinemia as a possible factor in the activation of motor function of the duodenum in pancreatic atrophy in rats]

The effect of pancreas atrophy on myoelectrical activity of the duodenum and serum trypsin, trypsin inhibitor, amylase, lipase activity has been described. The activation of the duodenum motor activity has been established. The changes in the motor activity were connected with trypsin and trypsin inhibitor activity of serum. The motor activity changes were compensated by trypsin therapy.     

Effect of light in the control of growth by auxin and its inhibitor(s) in the sunflower

Diffusates from the hypocotyl and leaves of sunflower seedlings ( Helianthus annuus L. cv. Mammoth) contain both auxin and inhibitor(s) of auxin-induced growth. The auxin activity has been evaluated with the conventional Avena coleoptile bioassay employing negative curvature, The inhibitor activity has been determined with a newly developed bioassay, measuring the positive curvature response of the Avena coleoptile. This bioassay has been standardized by the response to 2,3,5-triiodo-benzoic acid. Diffusates from plants in darkness have higher auxin activity and lower inhibitor activity than diffusates from plants in light. Irradiation at 730 nm promotes auxin synthesis in leaves, and! irradiation at 660 nm promotes synthesis of the inhibitor.     

Affinity chromatography of a cysteine proteinase inhibitor from chicken egg protein

A method of affinity chromatography of the inhibitor of cysteine proteinases from chick egg protein using immobilized ficin has been developed. This method yields a highly active inhibitor in an essentially homogeneous state. The molecular weight of the inhibitor is 14,000. The inhibitor suppresses the activity of ficin and papain but produces no effect on the proteolytic activity of trypsin, chymotrypsin, Asp. oryzae serine proteinase or subtilisine. Isoelectric focusing of the inhibitor has revealed the major band with pI 4.35.     

A crystalline protein-proteinase inhibitor from pinto bean seeds.

A crystalline protein-proteinase inhibitor has been isolated from seeds of Pinto bean (Phaseolus vulgaris cultvar. Pinto). It has an average molecular weight of 19 000 as estimated by gel filtration. This crystalline inhibitor is highly active against both bovine pancreatic trypsin and alpha-chymotrypsin. Complexes of both trypsin-inhibitor and alpha-chymotrypsin-inhibitor have been isolated. The inhibitor which was derived from the dissociated trypsin-inhibitor complex was only 62% as effective as the original compound against either enzyme. In contrast, the inhibitor obtained from alpha-chymotrypsin-inhibitor complex retained its full original inhibitory activity for trypsin, but only 25% of its original activity against alpha-chymotrypsin. The dissociated inhibitor from alpha-chymotrypsin-inhibitor compex, despite its full inhibitory activity, had been modified to such an extent that it could no longer form any precipitable complex with trypsin. The crystalline protein-proteinase inhibitor is not homogeneous and has been resolved into two distinct inhibitors in terms of their physical and chemical properties. These two inhibitors are designated as Pinto bean proteinase inhibitor I and II and their respective minimum molecular weights are 9100 and 10 000. They differ most strikingly in their amino acid composition in that inhibitor II is void of both valine and methionine.     

Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide alpha 2,6-N-acetylneuraminyltransferase (EC 2.4.99.-).

The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity.     

Detection of RNAase inhibitor from different species and organs

Human placental alkaline RNAase inhibitor was purified to homogeneity. Activity was measured after each purification step. The final identification of the purified protein was done by two-dimensional polyacrylamide gel electrophoresis and by immunoblotting. Antibodies were prepared by immunization of rabbits with the highly purified inhibitor. The availability of the antiserum directed against the human inhibitor enabled the detection of RNAase inhibitor from various other organs and species. This procedure has the advantage over the usual activity test in that the inhibitor can be found even if its activity has been lost.     

A new mechanism of regulation of rat liver acetyl-CoA carboxylase activity

A factor has been found in rat liver supernatant solution which inhibits acetyl-CoA carboxylase activity regardless of the presence or absence of Mg2+ and ATP. Inactivation of the enzyme has been demonstrated via radiochemical and spectrophotometric assay procedures. The inactivation of acetyl-CoA carboxylase is not attributable to either malonyl-CoA decarboxylase activity, to phosphorylation of the enzyme, or to action on substrates or cofactors of the reaction. The activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin. Dialyzing the inhibitor for 24 h at 4 degrees C does not alter its activity in inhibiting acetyl-CoA carboxylase. Hence, it appears that the inhibitor is a regulatory protein that acts directly on acetyl-CoA carboxylase.     

Inhibitor of uracil-DNA glycosylase induced by bacteriophage PBS2. Purification and preliminary characterization

A PBS2 phage-coded inhibitor of uracil-DNA glycosylase activity from Bacillus subtilis has been purified extensively and characterized preliminary. The inhibitor has a relative S value of 1.44 +/- 0.08 measured by sedimentation in 15 to 40% glycerol density gradients. It is unusually stable to heating and to the presence of sodium dodecyl sulfate and/or 8 M urea. The inhibitor has no known cofactor requirement and is active in the presence of 10 mM EDTA. Inhibitor activity is sensitive to digestion with proteinase K, but is insensitive to DNase or RNase digestion. The purified inhibitor behaves anomalously during electrophoresis in poly-acrylamide gels containing sodium dodecyl sulfate; however, experiments designed to show that the inhibitor is a glycoprotein were negative. The inhibitor clearly contains a protein required for activity, however, the possibility that some other molecular component is part of the active inhibitor cannot be excluded.     

Interferon inhibitor in the blood of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) patients at advanced stages of the disease have an interferon inhibitor in the blood circulation. This inhibitor can block antiviral activity of all three types of human interferons and can significantly reduce the synthesis of interferon alpha by the treated lymphocytes obtained from normal healthy individuals. Available evidence suggests that inhibitor activity is neither because of the antibody to interferon nor due to high level of protease-like activity in the plasma. The inhibitor has also been shown to be effective in eliminating the interferon-mediated enhancement of natural killer cell activity. Interferon inhibitory activity was not detected in any of the sera taken from normal healthy individuals. Identification and characterization of interferon inhibitor has direct bearing upon effective utilization of interferons in the clinic.     

Some aspects of pyridoxal phosphokinase in chick brain

Abstract— The activity of pyridoxal phosphokinase (EC 2.7.1.35) has been studied in two brain areas of the White Leghorn chick during post-hatch development. Activities of this enzyme were approximately the same in both forebrain and cerebellum at 2 days of age but when maximum activity was reached, at 20-25 days, the enzyme activity in forebrain was considerably higher than in the cerebellum. In homogenates, the activity of pyridoxal phosphokinase (as measured by pyridoxal phosphate formation) was inhibited by a particulate-bound inhibitor. In the forebrain of the 15-day-old chick, this inhibitor was detected in concentrations 3- to 5-fold greater than in the cerebellum. The inhibitor appeared to be an atypical ATPase which approached adult levels in the chick forebrain by two days after hatching. The possible physiological significance as well as the possible artifactual nature of this pyridoxal phosphokinase inhibitor in the in vitro assay system has been considered.     

Cissus quadrangularis extract enhances biomineralization through up-regulation of MAPK-dependent alkaline phosphatase activity in osteoblasts

Cissus quadrangularis Linn. has been implicated as therapeutic agent for enhancing bone healing. Though its osteogenic activity has been suggested, the underlying mechanism still remains unclear. In the present study, the effects of ethanol extract of C. quadrangularis (CQ-E) on osteoblast differentiation and function were analyzed using murine osteoblastic cells. The results indicated that mRNA expressions of osteoblast-related genes were not affected by the CQ-E treatment. However, alkaline phosphatase (ALP) activity and the extent of mineralized nodules were significantly increased in treated cells compared with controls. The addition of an extracellular regulated kinase 1/2 inhibitor, a Jun N-terminal kinase 1/2/3 inhibitor and a p38 mitogen-activated protein kinase (MAPK) inhibitor resulted in significantly decreased ALP activity, preferentially by p38 MAPK inhibitor. These results suggested that CQ-E may regulate osteoblastic activity by enhancing ALP activity and mineralization process, and the increased ALP activity effect of CQ-E is likely mediated by MAPK-dependent pathway.     

Effect of pentoxyphylline on certain indicators of the proteinase-proteinase inhibitor system in rats upon administration of cycloheximide

The pentoxifylline influence on neutral proteinase, alpha-2-macroglobulin, trypsin-alpha-1-proteinase inhibitor and elastaseinhibitory activity under cycloheximide injection has been investigated. Two hours after cycloheximide injection the activity of neutral proteinases increases in rats serum, lungs, heart, liver and kidneys. The preliminary injection of pentoxifylline prevents increase of neutral proteinases activity. Cycloheximide also decreases alpha-2-macroglobulin activity in serum and liver and trypsin-, elastaseinhibitory activity of alpha-1-proteinase inhibitor in all investigated organs. At using pentoxifylline the alpha-2-macroglobulin activity doesn't change in liver and increases in serum in comparison with only cycloheximide and there are no observed any alpha-1 inhibitor proteinase activity changes in rats serum and organs.     

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Incremental analysis: The application to quantitation of both enzyme activity and inhibitory activity in complex subcellular fractions

Single stage conventional analysis of neutral protease activity present in certain tumour cell cytoplasmic fractions has been demonstrated to be totally misleading due to hidden errors introduced by the complex interactions of the protease with a cytoplasmic inhibitor of this enzyme. A novel system has been developed which enables the rapid simultaneous quantitative analysis of neutral protease activity and inhibitor activity. This technique employs an insoluble fluorescein-labelled substrate (polymeric collagen fibrils) and relies on the sensitive fluorimetric assay of solubilised fluorescein-labelled peptides. This technique has been termed “incremental analysis” and the advantages of incremental analysis over conventional analysis methods have been described in detail. The experimentally obtained results can readily be resolved graphically or by simple computation.     

Inhibition of peptide initiation by a low molecular weight RNA from rabbit reticulocytes

A heat-stable inhibitor of protein synthesis has been isolated from the postribosomal supernatant of rabbit reticulocytes. Its activity is not susceptible to protease treatment but is destroyed by incubation with alkali. Inhibitory activity can be quantitatively recovered in the aqueous phase after phenol extraction and has the ultraviolet absorption spectrum of a nucleic acid. It is concluded that the inhibitor is RNA. The inhibitory activity sediments in the range of 3 S, but it has not been demonstrated whether the inhibitor RNA is a single molecular species. The inhibitory RNA does not affect peptide elongation but rather blocks a step of peptide initiation. It does not interfere with the formation of the ternary complex between initiation factor 2, GTP, and methionyl-tRNAMetf and does not activate a protein kinase phosphorylating initiation factor 2. The inhibitory RNA appears to be a novel type of RNA that inhibits polypeptide initiation at a step involving ribosomal subunits.     

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye)

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Developmental expression of a catalase inhibitor in maize

The expression of an endogenous catalase inhibitor has been studied during development of Zea mays. In the 3-day seedling, the inhibitor is expressed primarily in the scutellum and in the aleurone layer of the endosperm. These tissues also show the highest catalase activity at this stage. Inhibitor expression has also been studied temporally in the scutellum, roots, and shoot over the first 12 days of germination. Inhibitor expression shows an inverse relationship with catalase activity in the scutellum and in the shoot. The relationship is less rigid in the root, due probably to the low levels of inhibitor found in that tissue. The role of the inhibitor in catalase regulation is discussed.     

Enzyme inhibitors from plants. Isolation and characterization of a protease inhibitor from arrow root (Maranta arundinaceae) tuber

A protease inhibitor from arrow root (Maranta arundinaceae) tuber has been isolated in a homogeneous form. The inhibitor has a Mr of 11,000-12,000; it inhibited bovine trypsin, bovine enterokinase, bovine α-chymotrypsin and the proteolytic activity of human and bovine pancreatic preparations. The inhibitor is resistant to pepsin, and elastase. It could withstand heat treatment at 100°C for 60 min and exposure to a wide range of pH (1.0–12.5) for 72 h at 4°C without loss of activity. Arginyl groups are essential for the action of the inhibitor. Preincubation of the inhibitor at pH 3.7 with trypsin or chymotrypsin caused nearly a two-fold increase in inhibitor potency     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.