Biochemistry of human alpha amylase isoenzymes.

The purpose of this article is to review recent literature on the isoenzymes of alpha amylase. Although some studies are cited from the literature of fields other than clinical biochemistry, the aim is to bring together findings that may be of interest to clinical laboratory physicians and scientists. It is hoped that this will be useful in suggesting further studies of amylase. To this end, the review is more selective than exhaustive. The review will discuss the history and chemistry alpha amylases, the measurement of amylase and amylase isoenzymes, posttranslational modifications of human amylases, and the genetics of human pancreatic and salivary amylases. Finally, we will discuss other tissue sources of amylase with emphasis on "genital" amylases and their relationship to the amylase found in serous ovarian tumors.

Amylase from human serous ovarian tumors: purification and characterization.

Human serous-type ovarian tumors contain an acidic isoenzyme of amylase. Previous attempts at purification of tumor amylases have yielded preparations contaminated with other proteins. The purification scheme presented here incorporates an affinity-chromatography procedure, with use of cycloheptaamylose linked to epoxy-activated Sepharose, that is specific for alpha-amylase (EC 3.2.1.1). Purified amylase isoenzyme from a human serous ovarian tumor was characterized and compared with the purified salivary and pancreatic isoenzymes. All three were similar in amino acid composition, pH optimum, substrate specificity, calcium requirement, heat inactivation, and Km for maltotetraose substrate. The ovarian tumor amylase was similar to the salivary and distinct from the pancreatic enzyme by apparent molecular mass and doublet formation on sodium dodecyl sulfate--polyacrylamide electrophoresis, specific activity of pure enzyme, and sensitivity to specific alpha-amylase inhibitors. All three isoenzymes differed in net electrical charge as evidenced by diethylaminoethyl-Sephadex ion-exchange chromatography and isoelectric focusing. The tumor amylase is clearly distinct from the pancreatic and differs from the salivary enzyme in net electrical charge. Evidence is presented that this charge difference may reflect, at least in part, deamidation of an amylase that is similar to or identical with salivary amylase.

Amylase inhibits Neisseria gonorrhoeae by degrading starch in the growth medium.

Highly purified salivary alpha-amylase inhibited the growth of fresh isolates of Neisseria gonorrhoeae on GC agar base medium supplemented with 2% IsoVitaleX (BBL Microbiology Systems). Hydrolysis of starch in the medium by amylase resulted in a negative starch-iodine test. However, purified amylase did not inhibit gonococcal growth on agar plates that contained hemoglobin (chocolate agar). This effect was not caused by inhibition of amylase, since amylase activity was the same in the presence or absence of blood products. Moreover, survival of N. gonorrhoeae in buffered saline was not affected by amylase. These results suggest that amylase inhibited the growth of N. gonorrhoeae on GC agar base plates by hydrolyzing starch.

Improved DEAE-Sephadex column chromatography in measuring amylase in serous ovarian neoplasms, and results for 13 cases.

We describe a column-chromatographic method for measuring amylase activity in cyst fluids of serous ovarian tumors. Using this technique, we confirm and extend the previous report of an "acidic" amylase in serous ovarian tumor cyst fluids. This form of the enzyme was eluted from DEAE-Sephadex mini-columns with a high-salt buffer. It accounted for 45 to 100% of the amylase in 13 cyst fluids. This "high-salt" amylase was also present in the tumor tissues. The acidic nature of the amylase does not appear to be due to sialic acid residues, because the chromatographic behavior of the amylase is unaffected by treatment with neuraminidase. We conclude that the "acidic" amylase reported previously in two serous ovarian tumors is a constant feature of these tumors and that it is distinct from the sialylated tumor amylase described by others.

Role of matrix protein in assembling the membrane of vesicular stomatitis virus: reconstitution of matrix protein with negatively charged phospholipid vesicles.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reconstituted into phospholipid vesicles by detergent dialysis. Reconstitution of the positively charged M protein occurred only in the presence of negatively charged phospholipids such as phosphatidylserine, phosphatidic acid, or phosphatidylinositol. Preformed vesicles containing negatively charged phospholipids also bound free M protein. Derivatization of the positively charged lysines in M protein with acetic anhydride or succinic anhydride prevented M protein reconstitution but did not affect the biological property of M protein to inhibit in vitro VSV transcription. An additional indication of the electrostatic nature of the M protein binding to the vesicles was that M protein could not be reconstituted in the presence of 0.5 M NaCl. Nonelectrostatic forces also appear to be involved in the association of the M protein with vesicles, since previously reconstituted M protein remained associated with the vesicles upon subsequent exposure to 0.5 M NaCl.

Localization of membrane-associated proteins in vesicular stomatitis virus by use of hydrophobic membrane probes and cross-linking reagents.

The location of membrane-associated proteins of vesicular stomatitis virus was investigated by using two monofunctional and three bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [(125)I]5-iodonaphthyl-1-azide and [(3)H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both of these monofunctional probes labeled the glyco-protein G and matrix M proteins, but [(125)I]5-iodonaphthyl-1-azide also labeled the nucleocapsid N protein and an unidentified low-molecular-weight component. Protein labeling of intact virions was unaffected by the presence of cytochrome c or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents, tartryl diazide, dimethylsuberimidate, and 4,4'-dithiobisphenylazide, were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [(3)H]palmitate. Only G and M proteins of intact virions were labeled with (3)H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome c but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked (3)H-phospholipid to both G and M protein. In contrast, the hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein, whereas the hydrophobic 4,4'-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. These data support the hypothesis that the G protein traverses the virion membrane and that the M protein is membrane associated but does not penetrate very deeply, if at all.

Purification and properties of rat liver mitochondrial glutathione peroxidase.

Glutathione peroxidase (glutathione:hydrogen peroxide oxidoreductase, EC 1.11.1.9) was purified from rat liver mitochondria. The enzyme was shown to be pure by polyacrylamide-gel electrophoresis and to contain multiple forms that differed in charge. Selenium was specifically associated with the enzyme. The enzyme was inhibited by iodoacetic acid and iodoacetamide in an unusual pattern of reduction by sulfhydryl compounds and pH dependency. The mitochondrial and cytoplasmic forms of the enzyme were compared, and an explanation of the inhibition patterns is offered.

Identification of the catalytic site of rat liver glutathione peroxidase as selenocysteine.

A procedure was developed to isolate 75Se-labeled rat liver glutathione peroxidase (glutathione:H2O2 oxidoreductase, EC 1.11.1.9) at 30--50% purity with 20--30% yields in 4--5 days. Using these preparations of glutathione peroxidase, the selenium moiety in the enzyme was identified as selenocysteine by derivatizing the seleno group with either iodoacetate or ethylenimine in the intact protein, hydrolyzing the protein with 6 N HCl, and cochromatographing the 75Se-labeled products with known standards. Techniques employed were anion-exchange chromatography, cation-exchange chromatography, gel-permeation chromatography, two-dimensional thin-layer chromatography, and automated amino acid analysis. The selenocysteine moiety was identified as the catalytic site in glutathione peroxidase by specifically labeling the enzyme with [14C]iodoacetate on the 75Se-labeled selenium atom and fractionating the 14C, 75Se-labeled derivative after acid hydrolysis. It was concluded that the reduced form of glutathione peroxidase contains the selenocysteine selenol (-SeH) at the catalytic site.