Purification and properties of an alpha-amylase inhibitor from wheat.

Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.

Isolation and characterization of four inhibitors from wheat flour which display differential inhibition specificities for human salivary and human pancreatic alpha-amylases.

Four alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors were isolated from an albumin fraction of wheat flour by ion-exchange and gel-filtration chromatography. The purified inhibitors were characterized according to their electrophoretic mobilities, molecular weights, carbohydrate, content, sulphydryl content, susceptibility to proteolytic digestion and specificities in inhibiting human salivary and pancreatic alpha-amylases. The properties of these inhibitors ae compared to similar proteins isolated by other workers.

Interaction of human alpha-amylases with inhibitors from wheat flour.

The interaction of four purified alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors with human salivary and pancreatic alpha-amylases was investigated. The inhibitory activity of the four proteins towards salivary alpha-amylase was significantly increased by pre-incubation of the enzyme with inhibitor before adding substrate. This effect was not observed with the inhibition of pancreatic alpha-amylase by inhibitors 1 and 2. Inhibition of both amylases was affected to different degrees by incubating starch with inhibitor prior to the addition of enzyme. Maltose, at concentrations which only slightly affected amylase activity, prevented the inhibition of both enzymes by all four inhibitors. Gel filtration studies on salivary amylase-inhibitor mixtures showed the formation of EI complexes on a mol-to-mol ratio. A similar complex between pancreatic alpha-amylase and inhibitor 4 was observed, though complex formation between pancreatic alpha-amylase and the other inhibitors was not clearly demonstrated.

Three-dimensional structure of lung elastin demonstrated by scanning electron microscopy/stereo-pair images.

The aim of this study was to expose the inflated 3-D structure of lung elastin. Formic acid digestion followed by freeze-drying unveiled the lamellar framework. The 3-D structure of elastin was well preserved within the alveolar septa and ducts, as demonstrated by scanning electron microscopy/stereo-pair photography. Elastin fibers are seen in the alveolar septa, which are continuous with the lamellae. The removal of collagen fibers and cells by formic acid was visualised as a function of time: The optimum was 48 hours. Transverse sections still retained some collagen fibrils and partially digested cells in addition to elastin as shown by transmission electron microscopy (TEM). Formic acid digestion followed by critical point drying caused damage to the lamellar structures and they appeared to collapse. Sodium hydroxide digestion combined with freeze-drying did not preserve the 3-D lamellar structure of elastin, but converted it into flat ribbonlike bands. The main structures remaining following alkali treatment were identified by TEM as collagen fibrils well preserved in their original locations.

Microvasculature of the pancreas, liver, and kidney in cerulein-induced pancreatitis.

Edematous pancreatitis was induced in 12 male Sprague-Dawley rats using supramaximal doses of the cholecystokinin analogue cerulein (5 micrograms/kg per hour). The microvasculature of the pancreas, liver, and kidney was examined using scanning electron microscopy of microvascular corrosion casts in 12 test animals and four controls at intervals of 30 minutes, 1 hour, 2 hours, and 4 hours. Distortion of the pancreatic and hepatic microvasculature was seen as early as 30 minutes and progressed during the study period. The renal vasculature remained normal throughout. Light microscopic analysis revealed no morphologic abnormalities in the walls of the pancreatic, hepatic, or renal microvasculature. This study demonstrates that cerulein-induced pancreatitis is associated with marked distortion of the pancreatic and hepatic microvasculature; the abnormalities start early in the disease and progress during the study period.

Activity of serum alpha-amylases in cystic fibrosis.

A new method which uses a differential inhibitor to measure pancreatic and salivary type alpha-amylases (EC 3.2.1.1) was applied to serum samples from 46 cystic fibrosis (CF) patients (age range 4-14 years) and 50 controls of the same age group. The levels of pancreatic type amylase were lower in the CF patients (median 26.5 I.U./1) than the controls (median 81.5) (P less than 0.001). The results for salivary-type enzyme, however, did not support the previously reported finding of higher than usual levels in CF patients. This discrepancy is probably due to differences in analytical methods. It is felt that this procedure will be of value in the investigation of patients for cystic fibrosis and other pancreatic disorders.

Problems associated with the radioimmunoassay of serum trypsin.

A commercial trypsin radioimmunoassay (RIA) kit was used for its ability to measure trypsin bound to the serum protease inhibitors, alpha 2 macroglobulin (alpha 2 M) and alpha 1 anti-trypsin (alpha 1 AT). Only 20% of trypsin bound to alpha 2 M and 70% bound to alpha 1 AT was detected by the assay system. Recovery of trypsin added to human serum varied from 0 to 20%. Standard curves prepared from purified human cationic trypsin did not exhibit parallelism with the kit standard curves. Inclusion of horse serum in the standard solutions improved the parallelism observed. Immunoreactive trypsin (IRT) levels obtained for serum samples were found to vary considerably depending on the standard curve used to calculate the assay results. Lower IRT levels were observed when trypsin standards prepared in the absence of horse serum were used as reference.

Antigenic independence of some microbial urate oxidases.

Antisera were prepared to urate oxidase derived from three microbial species, a yeast (Candida utilis), a mold (Aspergillus flavus), and a bacterium (Bacillus fastidious). The antisera inhibited enyme activity to a limited extent. Cross-reaction studies with preparations of the enzyme from these and other species indicated that the microbial enzyme exhibits a high degree of antigenic independence. This appeared to be particularly true of the bacteria studied.

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.