Biography:

In the past Jerry L. Hedrick has collaborated on articles with Umbert A. Urch and David J. Gusseck. One of their most recent publications is The use of radioiodinated protein substrates for the assay of trypsin and the hatching enzyme from the amphibian Xenopus laevis. Which was published in journal Analytical Biochemistry.

More information about Jerry L. Hedrick research including statistics on their citations can be found on their Copernicus Academic profile page.

Jerry L. Hedrick's Articles: (16)

The use of radioiodinated protein substrates for the assay of trypsin and the hatching enzyme from the amphibian Xenopus laevis

AbstractAssays for a protease in tissue or cell extracts using synthetic substrates (esters and amides) are often compromised by the presence of nonspecific and non-protease-associated esterase and amidase activities. In addition, substrate specificity for some proteases is dependent on structural aspects of the substrate, so-called secondary specificity sites. The above limitations were present in our attempts to assay and isolate the hatching enzyme from embryos of Xenopus laevis. We developed an assay for the hatching enzyme using 125I-labeled fertilization envelopes, the natural substrate for this enzyme. Iodination was accomplished using lactoperoxidase. To test for the general usefulness of the assay and to compare this assay with previously employed ones, the trypsin-catalyzed hydrolysis of 125I-labeled hemoglobin was also studied.

Isolation and characterization of the hatching enzyme from the amphibian, Xenopus laevis☆

AbstractThe proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its 125I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn2+ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn2+ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.

The use of radioiodinated protein substrates for the assay of trypsin and the hatching enzyme from the amphibian Xenopus laevis

AbstractAssays for a protease in tissue or cell extracts using synthetic substrates (esters and amides) are often compromised by the presence of nonspecific and non-protease-associated esterase and amidase activities. In addition, substrate specificity for some proteases is dependent on structural aspects of the substrate, so-called secondary specificity sites. The above limitations were present in our attempts to assay and isolate the hatching enzyme from embryos of Xenopus laevis. We developed an assay for the hatching enzyme using 125I-labeled fertilization envelopes, the natural substrate for this enzyme. Iodination was accomplished using lactoperoxidase. To test for the general usefulness of the assay and to compare this assay with previously employed ones, the trypsin-catalyzed hydrolysis of 125I-labeled hemoglobin was also studied.

Isolation and characterization of the hatching enzyme from the amphibian, Xenopus laevis☆

AbstractThe proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its 125I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn2+ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn2+ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.

The use of radioiodinated protein substrates for the assay of trypsin and the hatching enzyme from the amphibian Xenopus laevis

AbstractAssays for a protease in tissue or cell extracts using synthetic substrates (esters and amides) are often compromised by the presence of nonspecific and non-protease-associated esterase and amidase activities. In addition, substrate specificity for some proteases is dependent on structural aspects of the substrate, so-called secondary specificity sites. The above limitations were present in our attempts to assay and isolate the hatching enzyme from embryos of Xenopus laevis. We developed an assay for the hatching enzyme using 125I-labeled fertilization envelopes, the natural substrate for this enzyme. Iodination was accomplished using lactoperoxidase. To test for the general usefulness of the assay and to compare this assay with previously employed ones, the trypsin-catalyzed hydrolysis of 125I-labeled hemoglobin was also studied.

The use of radioiodinated protein substrates for the assay of trypsin and the hatching enzyme from the amphibian Xenopus laevis

AbstractAssays for a protease in tissue or cell extracts using synthetic substrates (esters and amides) are often compromised by the presence of nonspecific and non-protease-associated esterase and amidase activities. In addition, substrate specificity for some proteases is dependent on structural aspects of the substrate, so-called secondary specificity sites. The above limitations were present in our attempts to assay and isolate the hatching enzyme from embryos of Xenopus laevis. We developed an assay for the hatching enzyme using 125I-labeled fertilization envelopes, the natural substrate for this enzyme. Iodination was accomplished using lactoperoxidase. To test for the general usefulness of the assay and to compare this assay with previously employed ones, the trypsin-catalyzed hydrolysis of 125I-labeled hemoglobin was also studied.

Isolation and characterization of the hatching enzyme from the amphibian, Xenopus laevis☆

AbstractThe proteolytic activity released at the time of Xenopus laevis embryo hatching, termed the hatching enzyme, was purified and characterized in terms of its physical and enzymatic properties. Using predominantly isoelectric focusing and preparative ultracentrifugation, the enzyme was purified 2200-fold over the starting crude hatching media. From disc gel electrophoretic experiments, the most highly purified form of the enzyme had two enzymatically active charge isomers present with molecular weights of 62,500. With time, the purified enzyme gave rise to a family of enzymatically active charge isomeric proteins. The enzymatic activity of hatching enzyme toward its 125I-labeled natural substrate, the fertilization envelope, was optimal at pH 7.7 and was ionic strength dependent. The enzyme was inhibited by Zn2+ and by EDTA. From inhibition by the site-specific reagents diisopropylfluorophosphate and phenylmethylsulfonylfluoride, we concluded that the enzyme was of the serine protease type, although its inhibition by Zn2+ and EDTA prevents a clear and unequivocal classification of the protease. This enzyme is different from the hatching enzymes reported in fish and echinoderms, on the basis of size, but it is similar to that described in Rana chensinensis on the basis of size and specificity.

A molecular approach to fertilization: I. Disulfide bonds in Xenopus laevis jelly coat and a molecular hypothesis for fertilization☆

AbstractThe jelly coat surrounding the egg of Xenopus laevis is dependent on the integrity of disulfide bonds for maintenance of its structure. This was proved by the jelly coat dissolving action of various disulfide bond-breaking reagents, by the rate of jelly coat solubilization as a function of pH, and by the appearance of free sulfhydryl groups due to ultraviolet photolysis of the jelly coat. From the presence of disulfide bonds in the jelly coat, it is postulated that sulfhydryl-disulfide bonds may be involved in the process of fertilization. Sperm penetration through egg integuments (jelly coat and vitelline membrane) may involve a lysin which utilizes a disulfide bond as a substrate. In addition, gamete fusion is visualized as occurring by a sulfhydryl-disulfide bond interchange. In view of the occurrence of disulfide bonds in egg integuments of many different organisms, this molecular hypothesis for fertilization may be generally applicable. The observation of jelly coat solubilization by mercaptans now makes possible a study of the macro-molecular composition of jelly coats and their role in fertilization.

Full paperHatching in the toad Xenopus laevis: Morphological events and evidence for a hatching enzyme☆

AbstractThe hatching process in embryos of the toad Xenopus laevis consists of two temporally distinct phases. In phase 1, the embryo escapes sequentially from the two outermost jelly layers, J3 and J2, and during phase 2 the embryo hatches from the last remaining jelly coat layer J1 and the fertilization envelope. Phase 1 hatching appears to be a physical process caused by water inbibition of jelly coat layer J1 and dynamic changes in the volume enclosed by the fertilization envelope. The combined turgor pressure ruptures jelly coat layers J3 and J2. The subsequent phase 2 hatching is a result of both physical and chemical processes. Phase 1 hatching exposes layer J1 to the medium which, in contrast to jelly layers J2 and J3 is partially soluble, and permits its gradual dissolution during Phase 2. The embryo secretes a proteolytic enzyme from the frontal region which partially digests the fertilization envelope; subsequent embryo movement ruptures the weakened envelope and completes the hatching process.

Research lettersSubunit structure of a cortical granule lectin involved in the block to polyspermy in Xenopus laevis eggs

AbstractThe cortical granule lectin of Xenopus laevis eggs is a large molecular mass glycoprotein involved in the post-fertilization block to polyspermy. We have investigated the subunit structure of this lectin and found that the native molecule contains 10–12 monomers, each of which has considerable charge and size heterogeneity due to glycosylated side chains. In addition, significant amino acid sequence homology is indicated by peptide mapping of subunits separated by isoelectric focusing.

Proteolysis of Xenopus laevis egg envelope ZPA triggers envelope hardening

AbstractThe egg envelope of most animal eggs is modified following fertilization, resulting in the prevention of polyspermy and hardening of the egg envelope. In frogs and mammals a prominent feature of envelope modification is N-terminal proteolysis of the envelope glycoprotein ZPA. We have purified the ZPA protease from Xenopus laevis eggs and characterized it as a zinc metalloprotease. Proteolysis of isolated egg envelopes by the isolated protease resulted in envelope hardening. The N-terminal peptide fragment of ZPA remained disulfide bond linked to the ZPA glycoprotein moiety following proteolysis. We propose a mechanism for egg envelope hardening involving ZPA proteolysis by an egg metalloprotease as a triggering event followed by induction of global conformational changes in egg envelope glycoproteins.

Full PapersIsolation and Characterization of Ovochymase, a Chymotrypsin-like Protease Released during Xenopus laevis Egg Activation

AbstractA chymotrypsin-like protease contained in the perivitelline space of unactivated Xenopus eggs is released during egg activation and appears to participate in vitelline envelope conversion. This 30-kDa protease, which we have termed ovochymase, was isolated from the exudate of activated eggs using a soy bean trypsin inhibitor-agarose affinity column. The column eluant contained only two proteins, the 30-kDa ovochymase plus a 78-kDa chymotrypsin-like proteolytic activity, The 78-kDa protease was not usually observed in fresh egg exudate samples and thus was activated during the purification process and may represent the proposed precursor of the 30-kDa protease. The 30- and 78-kDa proteases were separated by gel filtration HPLC or by SDS-PAGE. The N-terminal amino acid sequence of SDS-PAGE-isolated ovochymase was determined to be VVGGQQAAPR. This conserved amino acid sequence, plus active site specific inhibition and substrate specificity studies, places ovochymase in the serine protease I family of enzymes. A two-dimensional protease activity gel revealed that ovochymase is present as several isozymes with a wide range of pI's.

Chapter 12 Isolation of Extracellular Matrix Structures from Xenopus laevis Oocytes, Eggs, and Embryos

Publisher SummaryThe extracellular matrix (ECM) surrounding amphibian eggs is composed of jelly coat layers, the egg envelope, and the fibrous elements of the perivitelline space. The oviposited egg envelope provides an initially penetrable structure to the sperm which is subsequently modified by products released in the cortical reaction to a sperm-impenetrable structure in the block to polyspermy reaction. At fertilization the egg envelope is chemically modified and its macromolecular permeability properties changed. This permeability change (hardening) results in an osmotically driven envelope elevation, owing to the influx of water into the perivitelline space. The jelly after fertilization functions as a “sticky substrate” for the adherence of the zygote to objects in its surroundings, protects the zygote against physical damage, and also provides a microbiological barrier (bacteria are rarely found within the jelly coat layers). Thus, after fertilization the ECM protects the developing embryo and functions as a barrier for the chemical and biological regulation of the embryo environment. This protective function of the ECM persists until the embryo develops into a free swimming tadpole and hatches from the ECM. Thus, the chapter briefly discusses isolation of ECM structures from Xenopus laevis oocytes, eggs, and embryos.

ArticleGas-Phase Scrambling of Disulfide Bonds during Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Analysis

Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds.

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