Danica Dabich

Rapid method for determination of arginase activity in tissue homogenates

Abstract Arginase activity has been established by measurements of (a) a decrease in arginine concentration (1–4), (b) an increase in ornithine concentration (5), or (c) an increase in urea concentration (6–14). The major drawbacks of many of these methods have been delineated by Righetti et al. (13), who noted that only few of the procedures (3,8,12–14) are useful for measurements of arginase activity in tissue extracts or homogenates. Assays with crude enzyme preparations, other than the method of Gilboe and Williams (3) for arginine, require formation and isolation of a derivative of urea (8), removal of interferences in tissue extracts by precipitation (14), or time consumed in counting radioactive end products of the reaction (12,13). The thiosemicarbazide-diacetyl-monoxime-urea (TDMU) assay (15) obviates these additional steps and, as shown here, has the advantage of being a direct colorimetric procedure applicable to crude enzyme preparations.

A sensitive polyacrylamide disc gel method for detection of proteinases

Abstract To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.

Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts.

Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocyts. The enzyme activities are distinguishable my means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-L-lysine-chloromethyl ketone or L-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.

Transport of glucosamine (aldohexoses) by preimplantation mouse blastocysts

A carrier-mediated system for uptake of glucosamine (and other aldohexoses) was identified in preimplantation mouse blastocysts. With early and late blastocysts, the values for Km (525 +/- 82 and 651 +/- 137 microM, respectively) and V (0.29 +/- 0.02 and 0.29 +/- 0.04 pmol/h per ng protein, respectively) for glucosamine are statistically indistinguishable. The same carrier system, therefore, is operative at the two developmental stages. Glucosamine uptake was temperature sensitive and inhibited by structural analogues and phloretin.

Trypsin-like inhibitor activity in mouse uteri during early gestation and delayed implantation.

Summary Specific activities of tryptic-like inhibitors (TLI) in 27,000g supernatant solutions of mouse uterine homogenates were determined for the following conditions: nonpregnant, pregnant, and delayed implantation. Quantification of inhibitors was achieved by a fluorometric kinetic assay. Inhibitor levels in uteri from Day 4 pregnant and from delayed implantation mice were the same as non-pregnant uterine control values. A significant increase in inhibitor activity (P = 0.05) was found in uteri from Day 6 pregnant mice. This increased activity returned to control levels by Day 12 of pregnancy. Possible functions of TLI in regulating proteolytic activity associated with early development and implantation are discussed.

Transport of naturally occurring amino acids and alpha-amino isobutyric acid by normal and diapausing mouse blastocysts

Abstract Uptake of 14 C-labelled α-amino isobutyric acid and a mixture of naturally occurring amino acids by normal mouse preimplantation blastocysts was found to be twice that of delayed implantation blastocysts. With both groups of embryos concentration of the amino acids against a gradient occurred. The results may explain, in part, the lower amino acid incorporation rates found with diapausing vs. normal blastocysts and open the question of “metabolic dormancy” of the diapausing embryo.

The source of synovial fluid alkaline phosphatase.

Summary Agar-gel electrophoresis shows that synovia! fluid alkaline phosphatase migrates differently from that of serum but similar to the enzyme of fresh cartilage and bone extracts. It is concluded that the syn-ovial fluid enzyme is derived from the articulating cartilage and not from serum. Purification of the synovial fluid enzyme by a method involving chromatography on TEAE-cellu-lose leads to modification of the enzyme that is accompanied by a change in electrophoretic mobility but not in its rate of sedimentation in a sucrose gradient. Changes in mobility of phosphatases occurred as the result of aging cartilage extracts or treating synovial fluid with neuraminidase. Chymotrypsin and trypsin, which do not alter the mobility, inactivate the phosphatase of cartilage. We thank Dr. Ben Moffett and Miss Janice Ruffing for performing the histological studies.

Deglycosylation with trifluoromethanesulfonic acid differentially affects inhibitor activities of turkey ovomucoid

Turkey ovomucoid is an inhibitor of both trypsin and chymotrypsin. Treatment of this glycoprotein with trifluoromethanesulfonic acid in anisole resulted in time-dependent removal of carbohydrate and altered its biological activity. After 6 h of treatment the apparent molecular mass obtained by SDS-PAGE decreased from 38 to 30 kDa. Carbohydrate analyses indicated loss of 94% of original saccharide residues. The inhibitory activity of each domain was analyzed independently by comparing enzymic activity of trypsin and chymotrypsin in the absence of inhibitor to that preincubated in the presence of varying amounts of native or deglycosylated ovomucoid, respectively. The results demonstrated that removal of saccharides with trifluoromethanesulfonic acid differentially affects the inhibitor activities of turkey ovomucoid. Decreased inhibitory activity of the trypsin domain was observed with casein and benzoyl arginine ethyl ester as substrates. In contrast, enhanced inhibitory activity of the chymotrypsin domain was observed with benzoyl tyrosine ethyl ester and methyl-O-succinyl-Arg-Pro-Tyr-p-nitroanilide, good substrates for chymotrypsin, but not with casein.

Progesterone induced diminished incorporation of exogenous14C-amino acids in diapausing mouse blastocysts

Under identical in vitro labeling conditions, significantly lower amounts of radioactivity were found a) in proteins of early diapausing blastocysts (days 7–9 p.c.) vs normal, late blastocysts (day 5 p.c.) and b) in proteins of days 19–23 p.c. diapausing blastocysts from ovariectomized mothers treated with progesterone vs similar embryos from untreated mothers. Thus, progesterone, which maintains the viability of embryos during prolonged diapause, causes diminished utilization of exogenous amino acids for protein synthesis in these embryos.

An Inhibitor of Trypsin-Like Activity in Rat Liver∗

Summary Inhibitors of lysosomal and tryptic hydrolysis of benzoyl-dl-arginine-β-naphthylamide (BANA) were shown to be present in the microsomal and 100,000g supernatant fractions of rat liver homogenates. The 100,000g soluble inhibitor was shown to be: (1) capable of inhibiting tryptic digestion of casein and BANA; (2) insoluble at pH 4.0 but soluble at neutral or basic pH values; (3) nondialyzable; and (4) heat-stable. The inhibitor was maximally active at pH 7.4 and above during lysosomal hydrolysis of BANA.