Aminotransferases extraction

Numerous additional instances can be cited where small, moderate, or considerable PLP stimulations of plant aminotransferases have been observed. In most of these cases, however, no absolute PLP dependence has been seen unless the enzyme was specially treated to remove the cofactor. Where PLP dependence is not absolute, stimulation resulting from a nonspecific binding or stabilizing effect cannot be entirely ruled out (cf. Lu and Mazelis, 1975). The variability of the response to PLP is noteworthy but largely unexplained. Recent evidence (Achituv and Bar-Akiva, 1976) suggests a possible nutritional basis for this variability. Glutamateroxaloacetate aminotransferase extracted from phosphate-deficient citrus plants exhibited PLP dependence in crude extracts, whereas the enzyme from non-deficient plants contained adequate amounts of endogenous PLP and showed no further PLP stimulation. 

A similar activity level was obtained in the deoxycholate, Triton X-100, and NP-40 extract preparations. Octyl glucoside and CHAPS extract preparations showed no detectable prephenate aminotransferase activity. When the hemoglobin step was used, there was no increase in the soluble activity recovered in the initial supernatant fraction, but the specific activity of the deoxycholate (the only detergent tried in this experiment) extract increased about tenfold. We would anticipate equally good results with use of Triton X-100 or NP-40 in combination with the hemoglobin step. 

Fig. (1). Effects of Extract of Abri Herba in Mice Treated with CC14 Effects of Soyasaponin I (Soya I), total OG-I (OG-I) and total OG-II (OG-II) on CC14 induced liver injury by oral administration. These doses were each 500mg/kg. Each column represents mean of 10 mice. p<0.05, p<0.01. AST (aspartate aminotransferase), ALT (alanine aminotransferase)...

Glucocorticoids also increase the activity of transaminases (aminotransferases), especially in the skeletal muscle. Aminotransferases serve to transfer the amino groups from amino acids to be metabolized to a-keto acids, especially pyruvate. In the latter case, the alanine thus formed is transported from the muscle into the bloodstream and extracted from there by the liver. In the liver, alanine is converted to glucose, and glucose may then return to the muscle as it does in the Cori cycle (Figure 18.4). This is the alanine cycle, and more about this is discussed in Chapter 20. Branched-chain amino acids are the principal donors of nitrogen to pyruvate in the muscle and are thus important actors in the alanine cycle. 

Sterri, S.H., Fonnum, F. (1978). Isolation of organic anions by extraction with liquid anion exchangers and its application to micromethods for acetylcholinesterase and 4-aminobutyrate aminotransferase. Fur. J. Biochem. 91 215-22. 

The activity of BCAA aminotransferase is low in liver and relatively high in skeletal muscle. The opposite is true of the activity of BCKA dehydrogenase. This difference in enzyme activity is most likely the cause of the small extraction of BCAA by the liver. The majority of BCAA is taken up by skeletal muscles and converted to their respective branched-chain keto acids, which are partly consumed in the muscle cells. The remainder of BCKA is released into the blood. Part of the circulating BCKA is extracted by the liver. Inside the hepatocytes, BCKA is either degraded in the oxidative metabolic pathway or reaminated to preserve the total body content of BCAA (Harper et al., 1984). 

Chart 1 illustrates our experimental results of "Pharmacological Effect of Puerariae Flos and Its Constituents" [12, 13]. The MeOH extract of this crude drug depresses various alcohol-induced unusual metabolisms, a decrease in ALT (Alanine aminotransferase) and AST (Aspartate aminotransferase), suppression of spontaneous movement, decrease in BG and TG, and a significant decrease in blood alcohol and acetaldehyde level. After separation of the MeOH extract, the obtained triterpenoidal saponin fr. (PF-SP) shows a decrease in ALT and AST, and TG and... 

In postmenopausal women taking 40 mg black cohosh extract daily for 4 months, no changes in total hepatic blood flow, bilirubin, y-glutamyltransferase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, serum albumin, or prothrombin time and concentration were observed (Nasr and Nafeh 2009). No changes in liver enzyme levels were observed in other studies with women taking 40 mg black cohosh daily for 3 or 4 months, or in women given single doses up to 128 mg (Bai et al. 2007 Osmers et al. 2005 van Breemen et al. 2009). 

No changes in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels were observed in rats orally administered 400 mg/kg of an alcohol extract of black horehound daily for 7 days (Nusier et al. 2007a). 

In a 21-day study, mice were given drinking water containing annatto extract (56 or 351 mg/kg) or the compound norbixin (0.8, 7.6, 66, or 274 mg/kg), and rats were given drinking water containing annatto extract (0.8, 7.5 or 68 mg/kg) or norbixin (0.8,8.5, or 74 mg/kg). In rats, no toxicity was detected by plasma chemistry. In mice, norbixin induced an increase in plasma alanine aminotransferase activity (Fernandes et al. 2002). 

In men with positive prostate biopsies, oral administration of a standardized green tea extract containing 1.3 g green tea polyphenols (800 mg EGCG) daily for 12 to 214 days (mean of 34 days) was studied a decrease in liver enzyme levels, including aspartate aminotransferase, alkaline phosphatase, and amylase, was observed (McLarty et al. 2009). 

In rats orally administered 2.5 or 5 g/kg of Pu-erh green or Pu-erh black tea extract daily for 28 days, alanine aminotransferase increased in males at the 5 g/kg dose, and creatinine increased in all animals at the 5 g/kg dose and in males at the 2.5 g/kg dose. Slight bile duct hyperplasia in the liver was also observed. No adverse effects were observed in animals treated with Pu-erh black tea extract (Wang et al. 2010). 

In rats orally administered 500 or 800 mg/kg of an ethanol extract of boldo or of the compound boldine daily for 90 days, increases in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and cholesterol were observed in the 800 mg/kg groups for both products after 30 or 60 days, but levels returned to normal after 90 days. A decrease in bilirubin was observed in the 800 mg/ kg groups for both products. No changes in liver enzymes or cholesterol levels were observed in the 500 mg/kg group (Almeida et al. 2000). 

In mice (average animal weight 187 g) intramuscularly administered 1 ml of a chloroform extract of quassia daily for 15 days, no changes in bilirubin, aspartate aminotransferase, alanine aminotransferase, or hemoglobin levels were observed (Parveen et al. 2003). 

Acute hepatitis mimicking iron overload syndrome was reported in a 35-year-old man who had been taking fo-ti (dose and duration unspecified). Laboratory studies included alanine transferase 2714 U/1 (normal <50 U/1), aspartate aminotransferase 1170 U/1 (normal <50 U/1), AP 137 U/1 (normal <130 U/1), total bilirubin 4.6 mg/dl (normal <1.4 mg/dl), direct bilirubin 3.0 mg/dl (normal <0.4 mg/dl), and ferritin 13,862 ng/ml (normal 8 to 282 ng/ml) and a fasting transferrin saturation of 86% (normal 20% to 60%). Analysis of the herbal supplement identified extracts from fo-ti including the anthraquinones emodin and physcion. The patient recovered after cessation of the herbal products, and liver function tests 4 months after hospitalization were normal (Laird et al. 2008). 

In a dosing study of Chinese salvia extract injections in patients with hepatitis B, doses of 8, 16, or 24 ml were administered for 60 days. Chinese salvia treatment was associated with a decrease in liver enzyme levels, including alanine aminotransferase and total bilirubin. All doses were apparently well tolerated, and no adverse events were reported in the English language abstract of this study (Ye et al. 2005). 

On the other hand, there is considerable positive evidence for PLP involvement in plant transaminases. Cruickshank and Isherwood (1958) found that partially purified wheat germ glutamate oxaloacetate aminotransferase was stimulated 50-80% by PLP whereas glutamaterpyruvate aminotransferase was not. However in mung bean mitochondria the reverse was observed (Bone and Fowden, 1960). Cauliflower glutamate oxaloacetate aminotransferase showed no PLP activation in crude extracts, but a two- to three-fold stimulation of activity was seen after ammonium sulfate fractionation. Pyridoxamine-P activated the latter enzyme much more slowly than PLP (Davies and Ellis, 1961). 

Kinetic data are available for many aminotransferases, though in most cases the enzyme preparations have consisted either of crude extracts or incompletely purified proteins. Examples of such data are shown in Table 1. The An, value for the keto acid substrate is usually lower than the for the amino acid substrate, but there are exceptions to this generalization. Observed An, values may depend on the conditions of assay, and especially on the ionic composition of the buffer employed. For animal enzymes the anionic components of the buffer are said to be of particular importance in affecting kinetic parameters (cf. Braunstein, 1973). Plant enzymes have not been very systematically investigated in this respect, but phosphate inhibition has been observed for a peroxisomal enzyme transaminating with glyoxylate (Rehfeld and Tolbert, 1972). 

Cruickshank and Isherwood (1958) established that wheat germ gluta-materoxaloacetate and glutamate pyruvate aminotransferase activities, which they did not separate, differed in their dependence on supplied PLP and in their response to inhibitors. These investigators therefore proposed that the two activities would turn out to be due to separate enzymes. A study of aspartate and alanine aminotransferase activity in crude pear tissue extracts resulted in a similar prediction (Romani, 1962). This prediction has subsequently been borne out many times in investigations with more highly purified enzymes (see Section III,I). 

The enzymes utilizing serine as amino donor and glyoxylate as amino acceptor are considered together here. Much attention has been paid to these aminotransferases, with the role of these enzymes in photorespiration being a particular focus of interest (see Keys, this volume. Chapter 9). Initial studies on impure extracts indicated the presence of glyoxylate aminotransferases which could use glutamate, aspartate, alanine, and serine as amino donors. The best donor to glyoxylate varied from one tissue to another (Cossins and Sinha, 1965). Leaf peroxisomes were subsequently shown to... 

Most plant aminotransferases are easily obtained in soluble form. Indeed, for many enzymes the bulk of the activity is recovered in the supernatant phase of tissue extracts. However, it has long been recognized that some of... 

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