Alpha-amylase Isoenzymes Research Articles

Analysis for alpha-amylase isoenzymes by automated isoelectric focusing.

We describe the fractionation of alpha-amylase (EC 3.2.1.1) isoenzymes by use of the Pharmacia "Phastsystem" electrophoresis apparatus. The separation is done by isoelectric focusing on "Phastgel IEF 5-8." A complete run, including staining, takes 4 h and can easily resolve eight isoenzymes. Samples can be analyzed in a routine laboratory with use of conventional reagents.

Hyperamylasemia and alpha-amylase isoenzymes in acute liver congestion due to cardiac circulatory failure.

Hyperamylasemia and alpha-amylase isoenzymes in acute liver congestion due to cardiac circulatory failure.

A monoclonal antibody that specifically inhibits human salivary alpha-amylase.

Our monoclonal antibody 88E8 specifically binds to and inhibits human salivary alpha-amylase (EC 3.2.1.1) and cross reacts negligibly with the pancreatic isoenzyme, inhibiting it by less than 1%, as compared with about 90% for the salivary isoenzyme. The antibody binds the S1 and S2 types of salivary alpha-amylase, but no pancreatic alpha-amylase isoenzyme forms. A pancreatic alpha-amylase assay involving 88E8 is under development, with alpha-glucosidase as auxiliary enzyme and p-nitrophenyl-maltoheptaoside as substrate; we give preliminary data on this assay. The assay has to be done by substrate start, because the antibody interacts very slowly with the enzyme in the presence of substrate. Assay results for pancreatic alpha-amylase correlate well with those for isoamylase assayed with use of an inhibitor from wheat-germ.

Mathematical treatment of data for calculating activities of alpha-amylase isoenzymes.

Methods for determining alpha-amylase isoenzymes by selective inhibition with a wheat-germ protein are practical and easy, and give accurate, precise results. The incomplete specificity of the inhibitory action is not a major drawback but does necessitate mathematical treatment of the data (i.e., enzymic activities measured before and after preincubation with the inhibitor) to ascertain the amount of the different isoamylases. Such an algorithm is quite simple and straightforward, because the isoenzymes can be calculated either arithmetically or geometrically, by using a linear standard curve, empirically obtained, that relates the fraction of activity remaining after inhibition (R/T) to the pancreatic isoenzyme fraction or to the percentage of total alpha-amylase (P/T or 100 X P/T). An alternative method, plotting R/T against the ratio of pancreatic to salivary isoenzyme (P/S), is inconvenient, necessarily yields a nonlinear curve, needlessly complicates the calculations, and has been a persistent source of confusion in many articles dealing with the differentiation of isoamylases.

Development of an agarose gel electrophoresis technique for determining alpha-amylase isoenzymes.

Evaluation of alpha-amylase isoenzymes as a clinical diagnostic aid by previous methodologies has been either too insensitive or too cumbersome for routine clinical laboratory use. With use of an agarose gel electrophoresis system (Worthington Diagnostic Systems, Inc.) serum amylase activity can easily be resolved into at least nine isoenzymes, the resolution being comparable with that of isoelectric focusing. Samples can be analyzed in less than one working day, with use of conventional reagents.

2-Chloro-4-nitrophenyl-beta-D-maltoheptaoside: a new substrate for the determination of alpha-amylase in serum and urine.

Tests for the determination of alpha-amylase activity based on oligosaccharides or their 4-nitrophenyl derivatives as substrates suffer from some disadvantages. Using a more acid indicator molecule and connecting this group in the beta-form to maltoheptaose leads to a new substrate, 2-chloro-4-nitrophenyl-beta-D-maltoheptaoside, for the determination of alpha-amylase activity. Due to its pK value, only half of the 4-nitrophenol formed from 4-nitrophenyl-alpha-D-maltoheptaoside is spectrophotometrically detectable, whereas the total product from the new substrate is responsible for a measurable absorbance. In addition the molar lineic absorbance of the product is 1.8 times higher. Thus the new test is more sensitive than those employing 4-nitrophenyl derivatives. Both P-type and S-type alpha-amylase isoenzymes have the same affinity for the new substrate; this is not the case for all the other substrates, i.e. oligosaccharides and their 4-nitrophenyl derivatives, used in alpha-amylase tests. The sample volume may be reduced, thereby removing interference by substances like bilirubin, glucose and haemoglobin. The test is insensitive to changes in temperature and pH and can be adapted to all mechanized analytical instruments.

Mechanism of action of human pancreatic and salivary alpha-amylase on alpha-4-nitrophenyl maltoheptaoside substrate.

We evaluated the enzymic mechanism by which alpha-4-nitrophenyl maltoheptaoside serves as a substrate for serum amylase (EC 3.2.1.1). Because polymeric substrates possess many potential sites of cleavage, the expression of enzymic activity as the number of micromoles of substrate transformed under defined conditions by an enzyme must be changed to "one microequivalent of group transformed." Therefore, we measured the activity of the isoenzymes of alpha-amylase with regard to suitable oligosaccharide substrates, which allowed us to express the catalytic activity in IUB units (U). By "high-performance" liquid chromatography we investigated the mechanism of human pancreatic and salivary alpha-amylase action, both alone and in combination with alpha-glucosidase (EC 3.2.1.20). On the basis of these results, we can describe exactly the entire reaction sequence and determine the stoichiometric coefficient of 4-nitrophenol within 0.02 mol/L (SD) produced under the assay conditions.

α-Amylase Synthesis in Wheat Kernels as Influenced by the Seed Coat

The effect of seed coat removal on the synthesis of alpha-amylase isoenzymes in wheat was investigated. The immature wheat endosperm-aleurone (seed coat and embryo detached) produced considerably less alpha-amylase activity than immature whole or de-embryonated wheat kernels, when incubated under identical conditions of 18.5 C and 99% humidity, in the presence or absence of gibberellic acid (GA(3)). The incubated endosperm-aleurone also exhibited unique alpha-amylase isoenzyme composition when compared to the isoenzyme compositions of incubated whole and de-embryonated immature and mature wheat kernels both in the presence or absence of GA(3). Subsequent studies indicated that the seed coat may contain factor(s) required for normal alpha-amylase isoenzyme synthesis.

The effect of gamma irradiation on the formation of alpha-amylase isoenzymes in germinating wheat

The biosynthesis of alpha-amylase during seedling growth commences after a prolonged lag-period in wheat, irradiated at a high dose (200 krad). Also, a different requirement for exogenous gibberellins (GA 3) to stimulate the enzyme synthesis is noted in control and irradiated seeds. Further, the developmental patterns of three major isoenzymes of alphaamylase (designated as α 1−, α 2 and α 3) during germination are different. It is observed that α 1-isoenzyme, which appears on the 4th day of germination in control, is delayed in its development and is undetectable up to 4 days in 200 krad samples. However, α 1-isoenzyme appears after 6 days or after 4 days in GA-treated samples in germinating seeds exposed to a high dose. These results suggest that two systems differing in their radiosensitivity and response to GA application are operating in germinating wheat for the synthesis of functional alpha-amylase molecules.

Electrophoretic amylase fractionation as an aid in diagnosis of pancreatic disease.

Six alpha-amylase (EC 3.2.1.1) isoenzymes have been resolved electrophoretically on cellulose acetate membranes in a discontinuous buffer system. The fastest migrating isoenzymes are of salivary origin (S1, S2, S3), the slower ones of pancreatic origin (P1, P2, P3). We determined the amylase isoenzyme distribution in the sera of 240 subjects. A specific pancreatic isoenzyme (P3) was observed in all clinically diagnosed cases of acute or chronic pancreatitis as well as in 15 of 40 renal-transplant patients. Moreover, P3 isoenzyme activity declined during apparent recovery from pancreatitis. The P2 isoenzyme appeared in 95% of all specimens, P1 in only 2%. The pancreatic isoenzymes were preferentially excreted in the urine of both renal-transplant patients and normal individuals. The major salivary isoenzyme, S1, was observed in 95% of all serum and urine samples; however, the S2 and S3 appeared less consistently. Our method is simple and rapid, and quite applicable for use in clinical evaluation of patients with pancreatitis or with certain nonpancreatic dysfunctions.

Embryoless Wheat Grain

Yorkstar wheat, grown in New York State, has a high percentage (10-11) of grains without embryos. The embryoless grains have viable aleurone layers and show no sign of injury. These grains are able to support alpha-amylase synthesis only in the presence of gibberellin A(3) (GA(3)). In the absence of GA(3) some protein synthesis occurs in embryoless grains during the early hours of soaking, indicating that such activity occurs prior to and independent of GA(3) induction of alpha-amylase. The level of beta-amylase on a dry weight basis is the same in embryoless and normal grains and decreases with time of soaking. In the presence of GA(3), beta-amylase decreases at a slower rate. Isoenzymes of alpha-amylase from GA(3)-treated embryoless and normal grains show quantitative as well as qualitative differences. Cycloheximide (60 mug/ml) completely inhibits the synthesis of alpha-amylase by embryoless grains. Of the RNA synthesis inhibitors, actinomycin D (60 mug/ml) was ineffective while 6-methylpurine (60 mug/ml) gave 65% inhibition without decreasing the number of isoenzymes.